Non-invasive systems and methods for selective activation of photoreactive responses

ABSTRACT

Products, compositions, systems, and methods for modifying a target structure which mediates or is associated with a biological activity, including treatment of conditions, disorders, or diseases mediated by or associated with a target structure, such as a virus, cell, subcellular structure or extracellular structure. The methods may be performed in situ in a non-invasive manner by application of an initiation energy to a subject thus producing an effect on or change to the target structure directly or via a modulation agent. The methods may further be performed by application of an initiation energy to a subject in situ to activate a pharmaceutical agent directly or via an energy modulation agent, optionally in the presence of one or more plasmonics active agents, thus producing an effect on or change to the target structure. Kits containing products or compositions formulated or configured and systems for use in practicing these methods.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional application62/038,674, filed Aug. 18, 2014 and is related to U.S. Ser. No.12/417,779 filed, Apr. 3, 2009, entitled “NON-INVASIVE SYSTEMS ANDMETHODS FOR IN-SITU PHOTOBIOMODULATION,” the entire contents of whichare hereby incorporated by reference. The present application is relatedto U.S. Provisional application Ser. No. 61/955,131, filed May 18, 2014,the entire contents of which are hereby incorporated by reference. Thepresent application is related to U.S. Provisional application Ser. No.61/331,990, filed May 6, 2010, and U.S. Provisional application Ser. No.61/443,019, filed Feb. 15, 2011, the entire contents of each of whichare hereby incorporated by reference. The present application is alsorelated to U.S. provisional patent application 61/161,328, filed Mar.18, 2009; U.S. provisional patent application 61/259,940, filed Nov. 10,2009; U.S. Provisional Applications Ser. No. 60/954,263, filed Aug. 6,2007, and 61/030,437, filed Feb. 21, 2008; U.S. application Ser. No.12/059,484, filed Mar. 31, 2008; U.S. application Ser. No. 11/935,655,filed Nov. 6, 2007; U.S. Provisional Applications Ser. No. 61/042,561,filed Apr. 4, 2008; 61/035,559, filed Mar. 11, 2008; and 61/080,140,filed Jul. 11, 2008; U.S. patent application Ser. No. 12/401,478 filedMar. 10, 2009; U.S. patent application Ser. No. 11/935,655, filed Nov.6, 2007; U.S. patent application Ser. No. 12/059,484, filed Mar. 31,2008; U.S. patent application Ser. No. 12/389,946, filed Feb. 20, 2009;and U.S. patent application Ser. No. 12/417,779, filed Apr. 3, 2009, theentire contents of each of which is hereby incorporated by reference.This application is related to U.S. patent application Ser. No.12/763,404 filed Apr. 20, 2010, the entire contents of which are herebyincorporated by reference. This application is related to U.S. patentapplication Ser. No. 12/843,188 filed Jul. 26, 2010, the entire contentsof which are hereby incorporated by reference. This application isrelated to U.S. patent application Ser. No. 12/891,466 filed Sep. 27,2010, the entire contents of which are hereby incorporated by reference.This application is related to U.S. patent application Ser. No.12/943,787 filed Nov. 10, 2010, the entire contents of which are herebyincorporated by reference. This application is related to U.S. patentapplication Ser. No. 13/054,279 filed Jul. 13, 2011, the entire contentsof which are hereby incorporated by reference. This application isrelated to U.S. patent application 61/505,849 filed Jul. 8, 2011, theentire contents of which are hereby incorporated by reference. Thisapplication is related to U.S. patent application Ser. No. 13/102,277filed May 6, 2011, the entire contents of which are hereby incorporatedby reference. This application is related to U.S. patent applicationSer. No. 13/204,355 filed Aug. 5, 2011, the entire contents of which arehereby incorporated by reference. This application is related to U.S.patent application 61/735,754 filed Dec. 11, 2012, the entire contentsof which are hereby incorporated by reference. This application isrelated to U.S. patent application 62/014,561 filed Jun. 19, 2014, theentire contents of which are hereby incorporated by reference. Thisapplication is related to U.S. patent application 61/792,125 filed Mar.15, 2013, the entire contents of which are hereby incorporated byreference. This application is related to U.S. patent application61/930,717 filed Jan. 23, 2014, the entire contents of which are herebyincorporated by reference. This application is related to U.S. patentapplication 61/955,131 filed Mar. 18, 2014, the entire contents of whichare hereby incorporated by reference. This application is related toU.S. patent application 61/955,547 filed Mar. 19, 2014, the entirecontents of which are hereby incorporated by reference. This applicationis related to U.S. patent application Ser. No. 14/103,084 filed Dec. 11,2013, the entire contents of which are hereby incorporated by reference.This application is related to U.S. patent application Ser. No.14/131,564 filed Jul. 11, 2014, the entire contents of which are herebyincorporated by reference. This application is related to U.S. patentapplication Ser. No. 14/206,337 filed Mar. 12, 2014, the entire contentsof which are hereby incorporated by reference. This application isrelated to U.S. patent application 62/018,915 filed Jun. 30, 2014, theentire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of Invention

The present invention relates to methods and systems for treating adisorder or condition in a subject.

Discussion of the Background

Photobiomodulation

Photobiomodulation also known as low level laser therapy (LLLT), coldlaser therapy, and laser biostimulation, is an emerging medical andveterinary technique in which exposure to low-level laser light canstimulate or inhibit cellular function leading to beneficial clinicaleffects. The “best” combination of wavelength, intensity, duration andtreatment interval is complex and sometimes controversial with differentdiseases, injuries and dysfunctions needing different treatmentparameters and techniques.

Certain wavelengths of light at certain intensities (delivered by laser,light emitting diode LED or another monochromatic source) will, forexample, aid tissue regeneration, resolve inflammation, relieve pain andboost the immune system. The exact mechanism is still being explored anddebated but it is agreed that the mechanism is photochemical rather thanheat-related. Observed biological and physiological effects includechanges in cell membrane permeability, and up-regulation anddown-regulation of adenosine triphosphate and nitric oxide.

Light-induced biological effects depend on the parameters of theirradiation (wavelength, dose, intensity, irradiation time, depth of atarget cell, and continuous wave or pulsed mode, pulse parameters).(See, e.g., Karu I T, Low-Power Laser Therapy”, in Biomedical PhotonicsHandbook, Vo-Dinh T. Ed., CRC Press, Boca Raton, Fla., pp. 48-1 to48-25, (2003)). Laser average power is typically in the range of 1-500mW; some high peak power, short pulse width devices are in the range of1-100 W with typically 200 ns pulse widths. The average beam irradiancethen is typically 10 mW/cm²-5 W/cm². The wavelength is typically in therange 600-1000 nm. The red-to-near infrared (NIR) region is preferredfor photobiomodulation. Other wavelengths may be also used, e.g., UVlight for neurons and green light for prostate tissue. Maximumbiological responses often occur when irradiated at 620, 680, 760, and820-830 nm (Karu T I, et al., (1998). The Science of Low Power LaserTherapy. Gordon and Breach Sci. Publ., London). Large volumes andrelatively deeper layers of tissues can be successfully irradiated bylaser only (e.g., inner and middle ear diseases, injured siatic oroptical nerves, inflammations). The LEDs are used for irradiation ofsurface injuries.

A photoacceptor first absorbs the light used for the irradiation. Afterpromotion of electronically excited states, primary molecule processesfrom these states can lead to a measurable biological effect (viasecondary biochemical reaction, or photosignal transduction cascade, orcellular signaling) at the cellular level. A photoacceptor foreukaryotic cells in red-to-NIR region is believed to be the terminalenzyme of the respiratory chain cytochrome c oxidase located in cellmitochondrion. In the violet-to blue spectra region, flavoprotein (e.g.,NADHdehydrogenase in the beginning of the respiratory chain) is alsoamong the photoacceptors.

Clinical applications of photobiomodulation include, for example,treating soft tissue and bone injuries, chronic pain, wound healing,nerve regeneration, sensory regeneration/restoration and possibly evenresolving viral and bacterial infections, treating neurological andpsychiatric diseases (e.g., epilepsy and Parkinson's disease) (e.g.,Zhang F., et al., Nature, 446:617-9 (Apr. 5, 2007; Han X., et al., PloSONE, 2(3):e299 (Mar. 21, 2007); Arany P R, et al., Wound Repair Regen.,15(6):866-74 (2007); Lopes C B, et al., Photomed. Laser Surg.,25(2):96-101 (2007)). One clinical application showing great promise isthe treatment of inflammation, where the anti-inflammatory effect oflocation-and-dose-specific laser irradiation produces similar outcomesas NSAIDs, but without the potentially harmful side-effects (Bjordal JM, Couppé C, Chow R T, Tuner J, Ljunggren E A (2003). “A systematicreview of low level laser therapy with location-specific doses for painfrom chronic joint disorders”. The Australian journal of physiotherapy49(2):107-16).

An NIR light treatment can prevent cell death (apoptosis) in culturedneurons (brain) cells (Wong-Reiley M T, et al., JBC, 280(6):4761-71(2005)). Specific wavelengths of light can promote cellularproliferation to the activation of mitochondria, the energy-producingorganelles within the cell via cytochrome c oxidase. An NIR treatmentcan augment mitochondrial function and stimulate antioxidant protectivepathways. The evidence that the NIR treatment can augment mitochondrialfunction and stimulate antioxidant protective pathways comes fromphotobiomodulation experiments carried out using a laboratory model ofParkinson's disease (PD) (cultures of human dopaminergic neuronal cells)(Whelan H., et. al., SPIE, Newsroom, pages 1-3 (2008)).

It has also been shown that light has both inductive and inhibitoryeffect on cell growth and division in a red tide flagellate, Chattonellaantique (Nemote Y., Plant and Cell Physiol., 26(4):669-674 (1985)).

When the excitable cells (e.g., neurons, cardiomyocites) are irradiatedwith monochromatic visible light, the photoacceptors are also believedto be components of respiratory chain. It is clear from experimentaldata (Karu, T. I., (2002). Low-power laser therapy. In: CRC BiomedicalPhotonics Handbook, T. Vo-Dinh, Editor-in-Chief, CRC Press, Boca Raton(USA)) that irradiation can cause physiological and morphologicalchanges in nonpigmental excitable cells via absorption in mitochondria.Later, similar irradiation experiments were performed with neurons inconnection with low-power laser therapy. It was shown in 80's that He—Nelaser radiation alters the firing pattern of nerves; it was also foundthat transcutaneous irradiation with HeNe laser mimicked the effect ofperipheral stimulation of a behavioral reflex. These findings were foundto be connected with pain therapy (Karu T I, et al., (2002)).

When photoacceptors absorb photons, electronic excitation followed byphotochemical reactions occurring from lower excitation states (firstsinglet and triplet) take place. It is also known that electronicexcitation of absorbing centers alters their redox properties. Untilyet, five primary reactions have been discussed in literature (Karu T I,et al., (2002)). Two of them are connected with alteration of redoxproperties and two mechanisms involve generation of reactive oxygenspecies (ROE). Also, induction of local transient (very short time)heating of absorbing chromophores is possible. Details of thesemechanisms can be found in (Karu T I, et. al., (2002); Karu T I, et al.,(1998). The Science of Low Power Laser Therapy. Gordon and Breach Sci.Publ., London).

Photobiological action via activation of respiratory chain is believedto be a general mechanism occurring in cells. Crucial events of thistype of cell metabolism activation are occurring due to a shift ofcellular redox potential into more oxidized direction as well as due toATP extra synthesis. Susceptibility to irradiation and capability foractivation depend on physiological status of irradiated cells: thecells, which overall redox potential is shifted to more reduced state(example: some pathological conditions) are more sensitive to theirradiation. The specificity of final photobiological response isdetermined not at the level of primary reactions in the respiratorychain but at the transcription level during cellular signaling cascades.In some cells, only partial activation of cell metabolism happens bythis mechanism (example: redox priming of lymphocytes).

Far red and NIR radiation have been shown to promote wound healing,e.g., infected, ischemic, and hypoxic wounds (Wong-Reley, W T T, JBC,280(6):4761-4771 (2005)). Red-to-NIR radiation also protects the retinaagainst the toxic actions of methanol-derived formic acid in a rodentmodel of methanol toxicity and may enhance recovery from retinal injuryand other ocular diseases in which mitochondrial dysfunction ispostulated to play a role (Eells J T., PNAS, 100(6):3439-44 (2003)).Other clinical applications of photobiomodulation is repair of soft andbone tissues by IR laser irradiation (Martinez M E, et al., Laser inMed. Sci., 2007). Invasive laser assisted liposuction is a recentlydeveloped method, wherein a laser fiber is introduced through a tubeinto the skin and directly to the fat cells causing the cells to raptureand drain away as liquid (Kim K H, Dermatol. Surg., 32(2):241-48(2006)). Tissue around the area is coagulated. Yet, another applicationof photobiomodulation is a non-surgical varicose vein treatment (anendovenous laser therapy), wherein a laser is threaded through anincision and the full length of the varicose vein (Kim H S, J. Vasc.Interv. Radiol., 18(6):811 (2007)). When the laser is slowly withdrawn,heat is applied to the vein walls, causing the vein to permanently closeand disappear.

Yet, another area of application of photobiomodulation is a directcontrol (or modulation) of brain cell activity with light. The techniqueis based upon NIR spectroscopy and is simpler to use and less expensivethan other methods such as functional magnetic resonance imaging andpositron emission tomography.

Photostimulation can be used to activate a light-sensitive protein suchas rhodopsin (ChR2), which can then excite the cell expressing theopsin.

It has been shown that channelrhodopsin-2, a monolithic proteincontaining a light sensor and a cation channel, provides electricalstimulation of appropriate speed and magnitude to activate neuronalspike firing. Recently, photoinhibition, the inhibition of neuralactivity with light, has become feasible with the application ofmolecules such as the light-activated chloride pump halorhodopsin toneural control. Together, blue-light activated channelrhodopsin-2 andthe yellow light-activated chloride pump halorhodopsin enablemultiple-color, optical activation and silencing of neural activity.

ChR2 photostimulation involves genetic targeting ChR2 to neurons andlight pulsing the neurons expressing ChR2 protein. The experiments havebeen conducted in vitro and in vivo in mice by in vivo deep-brainphotostimulation using optical fibers to deliver light into the lateralhypothalamus (Adamantidis A R, et al., Nature 450:420-425 (2007)).Genetic targeting of ChR2 allows exclusive stimulation of definedcellular subsets and avoids the need for addition of the cagedglutamate, facilitating photostimulation in vivo (Wang H., et al., PNAS,104(19):8143-48 (2007)). ChR2 photostimulation has been used forrestoring visual activity in mice with impaired vision, to evokebehavioral responses in worms and flies (Wang H., et al., 2007). Therobust associative learning induced by ChR2-assisted photostimulaiton inmice opens the door to study the circuit basis of perception andcognition in vivo (Huber D., et al., 2007). This kind of neuronaltargeting and stimulation might have clinical application, e.g., deepbrain stimulation to treat Parkinson's disease and other disorders,controlling behavioral, perceptional and cognitive characteristics, andfor imaging and studying how the brain works (Zhang F., et al., NatureMethods, 3(10):785-792 (2006); Wong-Riley M T., et al., JBC,280(6):4761-4771 (2005)).

Another gene, chloride pump (NpHR), which is borrowed from a microbecalled an archaebacterium, can make neurons less active in the presenceof yellow light. Combined, the two genes ChR2 and NpHR can now makeneurons obey pulses of light like drivers obey a traffic signal: Bluemeans “go” (emit a signal), and yellow means “stop” (don't emit).

Light-sensitive proteins can be introduced into cells or live subjectsvia a number of techniques including electroporation, DNAmicroinjection, viral delivery, liposomal transfection andcalcium-phosphate precipitation.

A third photostimulation technique is chemical modification of ionchannels and receptors to render them light-responsive. Some of the mostfundamental signaling mechanisms in a cell involve the release anduptake of Ca²⁺ ions. Ca²⁺ is involved in controlling fertilization,differentiation, proliferation, apoptosis, synaptic plasticity, memory,and developing axons. It has been shown that Ca²⁺ waves can be inducedby UV irradiation (single-photon absorption) and NIR irradiation(two-photon absorption) by releasing caged Ca²⁺, an extracellularpurinergic messenger InsP3 (Braet K., et al., Cell Calcium, 33:37-48(2003)), or ion channel ligands (Zhang F., et al., 2006).

Directly controlling a brain cell activity with light is a novel meansfor experimenting with neural circuits and could lead to therapies forsome disorders. This accomplishment is a step toward the goal of mappingneural circuit dynamics on a millisecond timescale to see if impairmentsin these dynamics underlie severe psychiatric symptoms.

In living organisms, scientists were able to cause worms, C. elegans, tostop swimming while their genetically altered motor neurons were exposedto pulses of yellow light intensified through a microscope. In someexperiments, exposure to blue light caused the worms to wiggle in waysthey weren't moving while unperturbed. When the lights were turned off,the worms resumed their normal behavior.

Meanwhile, in experiments in living brain tissues extracted from mice,the researchers were able to use the technique to cause neurons tosignal or stop on the millisecond timescale, just as they do naturally.Other experiments showed that cells appear to suffer no ill effects fromexposure to the light. They resume their normal function once theexposure ends.

The most direct application of an optical neuron control isexperimenting with neural circuits to determine why unhealthy ones failand how healthy ones work.

In patients with Parkinson's disease, for example, researchers haveshown that electrical “deep brain stimulation” of cells can helppatients. By allowing researchers to selectively stimulate or dampendifferent neurons in the brain, the light stimulation techniques couldhelp in determining which particular neurons are benefiting from deepbrain stimulation.

Another potential application involves simulating neural communications.Because neurons communicate by generating patterns of signals-sometimeson and sometimes off like the 0s and 1s of binary computer code-flashingblue and yellow lights in these patterns could compel neurons to emitmessages that correspond to real neural instructions. The ability toartificially stimulate neural signals, such as movement instructions,could allow doctors to bridge blockages in damaged spinal columns,perhaps restoring some function to the limbs of paralyzed patients.

A low-intensity laser light-oxygen cancer therapy is another applicationof photobiomodulation. The light-oxygen effect (LOE), which involvesactivation of or damage to biosystems by optical radiation at lowoptical doses by direct photoexcitation of molecular oxygen dissolved ina biosystem so that it is converted to the singlet state, i.e., byphotogeneration of molecular singlet oxygen from 02 dissolved in cells,similar to photodynamic effect (Zakharov S D, et al., QuantumElectronics, 29(12):1031-53 (1999)). It was shown that the He—Ne laserradiation destroys tumor cells in the presence or absence of thephotosensitiser. The LOE can be activated by small optical doses, whichare 4-5 orders of magnitude lower that those found if a comparison ismade with the familiar analogue in the form of the photodynamic effect(PDE).

Problems with LLLT, Cold Laser Therapy, and Laser Biostimulation

The laser systems currently used for biostimulation do not allowperforming photobiomodulation in a region deep within thick tissuewithout a surgical invasion. Laser therapy is mostly conducted insurface or near surface target cells and tissue because penetration ofUV and red-to-N IR radiation used for photobiomodulation andphotobiostimulaiton is no more than a few centimeters beneath thesurface of the skin. In addition, imaging and stimulation of brain cellsis mainly possible in thin brain slices, or a thin monolayer orsuspension of cells. For deeper tissue laser therapy in situ, a subjectundergoes various invasive surgical procedures, e.g., invasive insertionof a fiber via incisions into a fat layer or veins, implanting aradiation source in deep tissue, or implanting a glass window above thebarrel cortex (Huber D., et al., Nature, 451:61-66 (2007)). It isfurther well recognized that another problem associated with theexisting methods of photobiomodulation is in differentiation of normalcells from target cells.

Phototherapy

There are two main types of reactions in phototherapy:

-   -   (1) Type I reactions involve electrons and hydrogen atoms, which        are transferred between photo-active molecules (also called        photosensitizers) and substrates or solvent molecules. Oxygen        may participate in subsequent reactions: e.g., psoralens in        photopheresis and PUVA.    -   (2) Type II reactions involve singlet oxygen formation by energy        transfer from PA molecules in the lowest triplet state to oxygen        in the ground state: e.g., photodynamic therapy (PDT)

Photodynamic therapy (PDT) is a treatment modality that uses aphotosensitizing agent and laser light to kill cells. PDT is arelatively new light-based treatment, which has recently been approvedby the United States Food & Drug Administration (FDA) for the treatmentof both early and late-stage lung cancer. Other countries have approvedPDT for treatment of various cancers as well. Unlike chemotherapy,radiation, and surgery, PDT is useful in treating all cell types,whether small cell or non-small cell carcinoma. PDT involves treatmentof diseases such as cancer using light action on a special photoactiveclass of drugs, by photodynamic action in vivo to destroy or modifytissue [Dougherty T. J. and Levy J. G., “Photodynamic Therapy andClinical Applications”, in Biomedical Photonics Handbook, Vo-Dinh T.,Ed., CRC Press, Boca Raton Fla. (2003)]. PDT, which was originallydeveloped for treatment of various cancers, has now been used to includetreatment of pre-cancerous conditions, e.g. actinic keratoses,high-grade dysplasia in Barrett's esophagus, and non-cancerousconditions, e.g. various eye diseases, e.g. age related maculardegeneration (AMD). Photodynamic therapy (PDT) is approved forcommercialization worldwide both for various cancers (lung, esophagus)and for AMD.

The PDT process requires three elements: (1) a PA drug (i.e.,photosensitizer), (2) light that can excite the photosensitizer and (3)endogenous oxygen. The putative cytotoxic agent is singlet oxygen, anelectronically excited state of ground state triplet oxygen formedaccording to the Type II photochemical process, as follows.PA+hν→ ¹PA*(S) Excitation¹PA*(S)→³PA*(T) Intersystem crossing for singlet to triplet state³PA*(T)+O₂→¹O*₂+PA Energy transfer from the drug to singlet oxygenwhere PA=photo-active drug at the ground state; ¹PA*(S)=excited singletstate; ³PA*(T)=excited triplet state; ¹O*₂=singlet excited state ofoxygen

Because the triplet state has a relatively long lifetime (μsec toseconds) only photosensitizers that undergo efficient intersystemcrossing to the excited triplet state will have sufficient time forcollision with oxygen in order to produce singlet oxygen. The energydifference between ground state and singlet oxygen is 94.2 kJ/mol andcorresponds to a transition in the near-infrared at ˜1270 nm. Most PAphotosensitizers in clinical use have triplet quantum yields in therange of 40-60% with the singlet oxygen yield being slightly lower.Competing processes include loss of energy by deactivation to groundstate by fluorescence or internal conversion (loss of energy to theenvironment or surrounding medium).

An important mechanism associated with PDT drug activity involvesapoptosis in cells. Upon absorption of light, the photosensitiser (PS)initiates chemical reactions that lead to the direct or indirectproduction of cytotoxic species such as radicals and singlet oxygen. Thereaction of the cytotoxic species with subcellular organelles andmacromolecules (proteins, DNA, etc) lead to apoptosis and/or necrosis ofthe cells hosting the PDT drug. The preferential accumulation of PDTdrug molecules in cancer cells combined with the localized delivery oflight to the tumor, results in the selective destruction of thecancerous lesion. Compared to other traditional anticancer therapies,PDT does not involve generalized destruction of healthy cells. Inaddition to direct cell killing, PDT can also act on the vasculature,reducing blood flow to the tumor causing its necrosis. In particularcases it can be used as a less invasive alternative to surgery.

There are several chemical species used for PDT includingporphyrin-based sensitizers. A purified hematoporphyrin derivative,Photofrin®, has received approval of the US Food and DrugAdministration. Porphyrins are generally used for tumors on or justunder the skin or on the lining of internal organs or cavities becausetheses drug molecules absorbs light shorter than 640 nm in wavelength.For tumors occurring deep in tissue, second generation sensitizers,which have absorbance in the NIR region, such as porphyrin-based systems[R. K. Pandey, “Synthetic Strategies in designing Porphyrin-BasedPhotosensitizers’, in Biomedical Photonics Handbook, Vo-Dinh T., Ed.,CRC Press, Boca Raton Fla. (2003)], chlorines, phthalocyanine, andnaphthalocyanine have been investigated.

PDT retains several photosensitizers in tumors for a longer time than innormal tissues, thus offering potential improvement in treatmentselectivity. See Corner C., “Determination of [3H]- and [14C]hematoporphyrin derivative distribution in malignant and normal tissue,”Cancer Res 1979, 3 9: 146-15 1; Young S W, et al., “Lutetium texaphyrin(PCI-0123) a near-infrared, water-soluble photosensitizer,” PhotochemPhotobiol 1996, 63:892-897; and Berenbaum M C, et al.,“Meso-Tetra(hydroxyphenyl)porphyrins, a new class of potent tumorphotosensitisers with favorable selectivity,” Br J Cancer 1986,54:717-725. Photodynamic therapy uses light of a specific wavelength toactivate the photosensitizing agent. Various light sources have beendeveloped for PDT, which include dye lasers and diode lasers. Lightgenerated by lasers can be coupled to optical fibers that allow thelight to be transmitted to the desired site. See Pass 1-11,“Photodynamic therapy in oncology: mechanisms and clinical use,” J NatlCancer Inst 1993, 85:443-456. According to researchers, the cytotoxiceffect of PDT is the result of photooxidation reactions, as disclosed inFoote C S, “Mechanisms of photooxygenation,” Proa Clin Biol Res 1984,170:3-18. Light causes excitation of the photosensitizer, in thepresence of oxygen, to produce various toxic species, such as singletoxygen and hydroxyl radicals.

Furthermore, when laser light is administered via external illuminationof tissue surfaces, the treatment effect of PDT is confined to a fewmillimeters (i.e. superficial). The reason for this superficiallimitation is mainly the limited penetration of the visible light usedto activate the photosensitizer. Thus, PDT is used to treat the surfacesof critical organs, such as lungs or intra-abdominal organs, withoutdamage to the underlying structures. However, even these treatmentsrequire significantly invasive techniques to treat the surface of theaffected organs. Clinical situations use the procedure in conjunctionwith surgical debulking to destroy remnants of microscopic or minimalgross disease. It is possible that the laser light and small amount ofremaining microscopic and minimal gross disease results in too little orhighly damaged structures.

Photopheresis has been successfully used for treatment of cellproliferation disorders. Exemplary cell proliferation disorders mayinclude, but are not limited to, cancer, bacterial infection, immunerejection response of organ transplant, solid tumors, viral infection,autoimmune disorders (such as arthritis, lupus, inflammatory boweldisease, Sjogrens syndrome, multiple sclerosis) or a combinationthereof, as well as aplastic conditions wherein cell proliferation islow relative to healthy cells, such as aplastic anemia. Of these, canceris perhaps the most well known.

Other successful application of PDT is, for example, cardiac ablasiontherapy, e.g., treating cardiac arrhythmias and atrial fibrillationwhich are believed to be a significant cause of cerebral stroke.

U.S. Pat. No. 6,811,562 describes administering a photoactivatable agentand subjecting cardiac tissue containing the administered agent to laserirradiation having a wavelength from 350 to 700 nm using invasivetechniques, e.g., a fiber optic element.

Yet, another application of PDT is photoangioplasty for arterialdiseases including de novo atherosclerosis and restinosis (Rockson A G,et al., Circulation, 102:591-596 (2000); Hsiang Y N., et al., J.Endovasc. Surg., 2:365-371 (1995)). In human clinical applications,endovascular light (730 nm) is delivered through a cylindrical fiberafter intravenous administration of motexafin lutetium. PDT is also usedfor preventing and treatment of intimal hyperplasia in blood vessels invivo (see, e.g., U.S. Pat. No. 6,609,014).

Age-related macular degeneration (AMD) is a cause of new blindness.Choroidal neovascularization leads to hemorrhage and fibrosis in anumber of ocular diseases. Conventional treatments utilize the argonlaser to occlude the leaking vessel by thermal coagulation. However, thepercentage of patients eligible for this treatment is limited. PDT isused for treating AMD and involves injecting verteporfin followed by theapplication of non-thermal light at 692 nm.

Improvement of clinical appearance of psoriatic plaques andpalmopustular psoriasis using PUVA with hematopotphyrin was firstreported in 1937. Acne, apopecia areata, portwine stains and hairremoval also show promise with PDT treatment.

In one existing treatment known as extracorporeal photopheresis (ECP),excellent results have been observed since its initial approval by theFDA in 1988.

Extracorporeal photopheresis (ECP) is a leukapheresis-basedimmunomodulatory therapy that has been approved by the US Food and DrugAdministration for the treatment of cutaneous T-cell lymphoma (CTCL).ECP, also known as extracorporeal photochemotherapy, is performed atmore than 150 centers worldwide for multiple indications. Long-termfollow-up data are available from many investigators that indicate ECPproduces disease remission and improved survival for CTCL patients. Inaddition to CTCL, ECP has been shown to have efficacy in the treatmentof other T-cell mediated disorders, including chronic graft versus hostdisease (GVHD) and solid organ transplant rejection. ECP use for thetreatment of autoimmune disease, such as systemic sclerosis andrheumatoid arthritis, is also being explored.

ECP is generally performed using the UVAR XTS Photopheresis Systemdeveloped by Therakos, Inc (Exton, Pa.). The process is performedthrough one intravenous access port and has 3 basic stages: (1)leukapheresis, (2) photoactivation, and (3) reinfusion, and takes 3-4hours to complete. A typical treatment session would resemble thefollowing sequence of events:

(1) One 16-gauge peripheral intravenous line or central venous access isestablished in the patient;

(2) Blood (225 mL) is passed through 3 cycles of leukapheresis, or 125mL of blood is passed through 6 cycles, depending on the patient'shematocrit value and body size. At the end of each leukapheresis cycle,the red blood cells and plasma are returned to the patient;

(3) The collected WBCs (including approximately 5% of the peripheralblood mononuclear cells) are mixed with heparin, saline, and8-methoxypsoralen (8-MOP), which intercalates into the DNA of thelymphocytes upon exposure to UVA light and makes them more susceptibleto apoptosis when exposed to UVA radiation;

(4) The mixture is passed as a 1-mm film through a sterile cassettesurrounded by UVA bulbs, resulting in an average UVA exposure of 2J/cm²; and

(5) The treated WBC mixture is returned to the patient.

Over the past 20 years, on-going research has explored the mechanism ofaction of ECP. The combination of 8-MOP and UVA radiation causesapoptosis of the treated T cells and may cause preferential apoptosis ofactivated or abnormal T cells, thus targeting the pathogenic cells ofCTCL or GVHD. However, given that only a small percentage of the body'slymphocytes are treated, this seems unlikely to be the only mechanism ofaction.

Other evidence suggests that ECP also induces monocytes to differentiateinto dendritic cells capable of phagocytosing and processing theapoptotic T-cell antigens. When these activated dendritic cells arereinfused into the systemic circulation, they may cause a systemiccytotoxic CD8⁺ T-lymphocyte-mediated immune response to the processedapoptotic T-cell antigens.

Finally, animal studies indicate that photopheresis may induceantigen-specific regulatory T cells, which may lead to suppression ofallograft rejection or GVHD.

Alternatively, a patient can be treated in vivo with a photosensitiveagent followed by the withdrawal of a sample from the patient, treatmentwith UV radiation in vitro (ex vivo), and re-injecting the patient withthe treated sample. This method is known for producing an autovaccine. Amethod of treating a patient with a photosensitive agent, exposing thepatient to an energy source and generating an autovaccine effect whereinall steps are conducted in vivo has not been described. See WO03/049801, U.S. Pat. Nos. 6,569,467; 6,204,058; 5,980,954; 6,669,965;4,838,852; 7,045,124, and 6,849,058. Moreover, the side effects ofextracorporeal photopheresis are well known and include nausea,vomiting, cutaneous erythema, hypersensitivity to sunlight, andsecondary hematologic malignancy. Researchers are attempting to usephotopheresis in experimental treatments for patients with cardiac,pulmonary and renal allograft rejection; autoimmune diseases, andulcerative colitis.

U.S. Pat. No. 5,829,448 describes sequential and simultaneous two photonexcitation of photo-agents using irradiation with low energy photonssuch as infrared or near infrared light (NRI). A single photon andsimultaneous two photon excitation is compared for psoralen derivatives,wherein cells are treated with the photo agent and are irradiated withNRI or UV radiation. The patent suggests that treating with a low energyirradiation is advantageous because it is absorbed and scattered to alesser extent than UV radiation.

Chen et al., J. Nanosci. and Nanotech., 6:1159-1166 (2006); Kim et al.,JACS, 129:2669-2675 (2007); U.S. 2002/0127224; and U.S. Pat. No.4,979,935 each describe methods for treatment using various types ofenergy activation of agents within a subject. However, each suffers fromthe drawback that the treatment is dependent on the production ofsinglet oxygen to produce the desired effect on the tissue beingtreated, and is thus largely indiscriminate in affecting both healthycells and the diseased tissue desired to be treated.

U.S. Pat. No. 6,908,591 describes methods for sterilizing tissue withirradiation to reduce the level of one or more active biologicalcontaminants or pathogens, such as viruses, bacteria, yeasts, molds,fungi, spores, prions or similar agents responsible, alone or incombination, for transmissible spongiform encephalopathies and/or singleor multicellular parasites, such that the tissue may subsequently beused in transplantation to replace diseased and/or otherwise defectivetissue in an animal. The method may include the use of a sensitizer suchas psoralen, a psoralen-derivative or other photosensitizer in order toimprove the effectiveness of the irradiation or to reduce the exposurenecessary to sterilize the tissue.

U.S. Pat. No. 5,957,960 describes a two-photon excitation device foradministering a photodynamic therapy to a treatment site within apatient's body using light having an infrared or near infrared waveband.

U.S. Pat. No. 6,235,508 describes antiviral applications for psoralensand other photoactivatable molecules. It teaches a method forinactivating viral and bacterial contaminants from a biologicalsolution. The method includes mixing blood with a photosensitizer and ablocking agent and irradiating the mixture to stimulate thephotosensitizer, inactivation of substantially all of the contaminantsin the blood, without destroying the red blood cells. The blocking agentprevents or reduces deleterious side reactions of the photosensitizer,which would occur if not in the presence of the blocking agent. The modeof action of the blocking agent is not predominantly in the quenching ofany reactive oxygen species, according to the reference.

U.S. Pat. No. 6,235,508 suggests that halogenated photosensitizers andblocking agents might be suitable for replacing 8-methoxypsoralen(8-MOP) in photopheresis and in treatment of certain proliferativecancers, especially solid localized tumors accessible via a fiber opticlight device or superficial skin cancers. However, the reference failsto address any specific molecules for use in treating lymphomas or anyother cancer. Instead, the reference suggests a process of photopheresisfor antiviral treatments of raw blood and plasma.

U.S. published application 2002/0127224 describes a method for aphotodynamic therapy comprising administering light-emittingnanoparticles and a photoactivatable agent, which may be activated bythe light re-emitted from the nanoparticles via a two-photon activationevent. An initiation energy source is usually a light emitting diode,laser, incandescent lamp, or halogen light, which emits light having awavelength ranging from 350 to 1100 nm. The initiation energy isabsorbed by the nanoparticles. The nanoparticles, in turn, re-emit lighthaving a wavelength from 500 to 1100 nm, preferably, UV-A light, whereinthe re-emitted energy activates the photoactivatable agent. Kim et al.,(JACS, 129:2669-75, 2/9/2007) describes indirect excitation of aphotosensitizing unit (energy acceptor) through fluorescence resonanceenergy transfer (FRET) from the two-photon absorbing dye unit (energydonor) within an energy range corresponding to 300-850 nm.

Psoralens and Related Compounds

U.S. Pat. No. 6,235,508 describes that psoralens are naturally occurringcompounds which have been used therapeutically for millennia in Asia andAfrica. The action of psoralens and light has been used to treatvitiligo and psoriasis (PUVA therapy; Psoralen Ultra Violet A). Psoralenis capable of binding to nucleic acid double helices by intercalationbetween base pairs; adenine, guanine, cytosine and thymine (DNA) oruracil (RNA). Upon sequential absorption of two UV-A photons, psoralenin its excited state reacts with a thymine or uracil double bond andcovalently attaches to both strands of a nucleic acid helix. Thecrosslinking reaction appears to be specific for a thymine (DNA) or auracil (RNA) base. Binding proceeds only if psoralen is intercalated ina site containing thymine or uracil, but an initial photoadduct mustabsorb a second UVA photon to react with a second thymine or uracil onthe opposing strand of the double helix in order to crosslink each ofthe two strands of the double helix, as shown below. This is asequential absorption of two single photons as shown, as opposed tosimultaneous absorption of two or more photons.

U.S. Pat. No. 4,748,120 of Wiesehan is an example of the use of certainsubstituted psoralens by a photochemical decontamination process for thetreatment of blood or blood products.

Additives, such as antioxidants are sometimes used with psoralens, suchas 8-MOP, AMT and I-IMT, to scavenge singlet oxygen and other highlyreactive oxygen species formed during photoactivation of the psoralens.It is well known that UV activation creates such reactive oxygenspecies, which are capable of seriously damaging otherwise healthycells. Much of the viral deactivation may be the result of thesereactive oxygen species rather than any effect of photoactivation ofpsoralens.

The best known photoactivatable compounds are derivatives of psoralen orcoumarin, which are nucleic acid intercalators. The use of psoralen andcoumarin photosensitizers can give rise to alternative chemical pathwaysfor dissipation of the excited state that are either not beneficial tothe goal of viral inactivation, or that are actually detrimental to theprocess. For psoralens and coumarins, this chemical pathway is likely tolead to the formation of a variety of ring-opened species, such as shownbelow for coumarin:

Research in this field over-simplifies mechanisms involved in thephotoactivation mechanism and formation of highly reactive oxygenspecies, such as singlet oxygen. Both may lead to damage of tumor cells,viruses and healthy cells. However, neither, alone or combined, lead toan auto vaccine effect. This requires an activation of the body's ownimmune system to identify a malignant cell or virus as threat and tocreate an immune response capable of lasting cytotoxic effects directedto that threat. It is believed, without being limiting in any way, thatphotoactivation and the resulting apoptosis of malignant cells thatoccurs in extracorporeal photophoresis causes the activation of animmune response with cytotoxic effects on untreated malignant cells.

Midden (W. R. Midden, Psoralen DNA photobiology, Vol I1 (ed. F. P.Gaspalloco) CRC press, pp. 1. (1988) has presented evidence thatpsoralens photoreact with unsaturated lipids and photoreact withmolecular oxygen to produce active oxygen species such as superoxide andsinglet oxygen that cause lethal damage to membranes.

U.S. Pat. No. 6,235,508 describes that 8-MOP and AMT are unacceptablephotosensitizers, because each indiscriminately damages both cells andviruses. Studies of the effects of cationic side chains on furocoumarinsas photosensitizers are reviewed in Psoralen DNA Photobiology, Vol. I,ed. F. Gaspano, CRC Press, Inc., Boca Raton, Fla., Chapter 2. U.S. Pat.No. 6,235,508 gleans the following from this review: most of the aminocompounds had a much lower ability to both bind and form crosslinks toDNA compared to 8-MOP, suggesting that the primary amino functionalityis the preferred ionic species for both photobinding and crosslinking.

U.S. Pat. No. 5,216,176 describes a large number of psoralens andcoumarins that have some effectiveness as photoactivated inhibitors ofepidermal growth factor. Halogens and amines are included among the vastfunctionalities that could be included in the psoralen/coumarinbackbone. This reference is incorporated herein by reference in itsentirety.

U.S. Pat. No. 5,984,887 describes using extracorporeal photopheresiswith 8-MOP to treat blood infected with CMV. The treated cells as wellas killed and/or attenuated virus, peptides, native subunits of thevirus itself (which are released upon cell break-up and/or shed into theblood) and/or pathogenic noninfectious viruses are then used to generatean immune response against the virus, which was not present prior to thetreatment. \

Problems with PDT

It is well recognized that a major problem associated with the existingmethods of diagnosis and treatment of cell proliferation disorders is indifferentiation of normal cells from target cells. Radiation therapyworks by irradiating cells with high levels of high energy radiationsuch as high energy photon, electron, or proton. These high energy beamsionize the atoms which make up a DNA chain, which in turn leads to celldeath. Unlike surgery, radiation therapy does not require placingpatients under anesthesia and has the ability to treat disorders deepinside the body with minimal invasion of the body. However, the highdoses of radiation needed for such therapies damages healthy cells justas effectively as it does diseased cells. Thus, similar to surgery,differentiation between healthy and diseased cells in radiation therapyis only by way of location. There is no intrinsic means for a radiationbeam to differentiate between a healthy cell from a diseased celleither. Another problem encountered in PDT therapy is the inability totreat target areas that are more than a few centimeters beneath thesurface of the skin without significant invasive techniques.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodfor the treatment of a condition, disorder or disease in a subject thatpermits treatment of a subject in any area of the body where thetreatment selectively activates plural biological responses depending ona selection of the wavelength of light generated internally or providedinternally within the body.

Thus, in one object of the present invention, there are provided aplurality of light emitters at different wavelengths corresponding torespective biological responses being activated by each of the differentwavelengths.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease in a subject which can useany suitable energy source as the initiation energy source to induce apredetermined change in a target structure in a subject in situ to treatthe condition, disorder or disease by way of the selective activationnoted above.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease using a modulation agentwhich adsorbs, intensifies or modifies the initiation energy into anenergy that effects a predetermined change in a target structure by wayof the selective activation noted above.

These and other objects of the present invention, which will become moreapparent in conjunction with the following detailed description of thepreferred embodiments, either alone or in combinations thereof, havebeen satisfied by the discovery of a method for treating a condition,disorder or disease in a subject, comprising:

applying an initiation energy from at least one source to a targetstructure in a subject in need of treatment, wherein the initiationenergy contacts the target structure and induces a predetermined changein said target structure in situ,

-   -   thus treating said condition, disorder or disease.

Yet a further object of the invention is further administer at least oneenergy modulation agent to said subject which adsorbs, intensifies ormodifies said initiation energy into an energy that effects apredetermined change in said target structure by way of the selectiveactivation noted above.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease which can use any suitableenergy source as the initiation energy source to activate theactivatable pharmaceutical agent and thereby cause a predeterminedchange in a target structure to treat a condition, disorder or diseaseby way of the selective activation noted above.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease using an energy cascade toactivate an activatable pharmaceutical agent that then treats cellssuffering from a condition, disorder or disease by way of the selectiveactivation noted above.

A further object of the present invention is to provide a method forgenerating an autovaccine effect in a subject, which can be in vivo thusavoiding the need for ex vivo treatment of subject tissues or cells, orcan be ex vivo by way of the selective activation noted above.

A further object of the present invention is to provide a method forgenerating an autovaccine effect in a subject, which can be in vivo thusavoiding the need for ex vivo treatment of subject tissues or cells, orcan be ex vivo by way of the selective activation noted above.

A further object of the present invention is to provide a computerimplemented system for performing the methods of the present invention.

A still further object of the present invention is to provide a kit anda pharmaceutical composition for use in the present invention methods.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 provides an exemplary electromagnetic spectrum in meters (1 nmequals 10⁻⁹ meters).

FIG. 2A and FIG. 2B are graphical representations of the depth ofpenetration of various wavelengths of energy into living tissue

FIG. 3 illustrates a system according to one exemplary embodiment of thepresent invention.

FIG. 3-1 illustrates an exemplary system according to one embodiment ofthe invention for producing a photo-reactive change in a medium.

FIG. 4 illustrates an exemplary computer implemented system according toan embodiment of the present invention.

FIG. 5 illustrates an exemplary computer system (1201) for implementingvarious embodiments of the present invention.

FIGS. 6A-6B are graphical representations of plasmonic nanostructuresand their theoretical electromagnetic enhancement at differentexcitation wavelengths.

FIGS. 7A-7G provide representative embodiments of plasmonicsphoto-active probes useful in the present invention.

FIGS. 8A-8B are graphical explanations of the plasmonics-enhanced effectof photospectral therapy used in the present invention.

FIGS. 9A-9J provide representative embodiments of plasmonics-activenanostructures.

FIG. 10 is a graphical representation of one embodiment of a PEPST probewith remote drug release.

FIGS. 11A-11C are graphical representations of several embodiments ofPEPST probes with various linkers for remote drug release.

FIGS. 12A-12G are graphical representations of several embodiments ofplasmonics photo-active probes with bioreceptors.

FIG. 13 is a graphical representation of the “therapeutic window” intissue and absorption spectra of biological components.

FIG. 14 is a graphical representation of an embodiment of the energymodulation agent(or excitation energy converter/EEC)-photo activator(PA) system of the present invention.

FIGS. 15A-15F are graphical representations of several embodiments ofplasmonics photo-active energy modulation agent-PA probes.

FIGS. 16A-16B show structures of various preferred embodiments of goldcomplexes exhibiting XEOL.

FIG. 17 shows the structure of a further embodiment of compoundexhibiting XEOL, namely a tris-8-hydroxyquinoline-aluminum complex.

FIG. 18 is a graphical representation of a plasmonics-enhanced mechanismfor a photo-active energy modulation agent-PA probe of the presentinvention.

FIG. 19 is a graph showing excitation and emission fluorescence spectraof psoralens.

FIGS. 20A-20C are graphical representations of an embodiment of a PEPSTenergy modulation agent-PA system with detachable bond.

FIG. 21 is a graphical representation of an embodiment of PEPST probesfor dual plasmonic excitation.

FIGS. 22A-22D are graphical representations of an embodiment of a use ofencapsulated photoactive agents.

FIGS. 23A-23B are simplified graphical representations of the use of thepresent invention principle of non-invasive PEPSI modality.

FIG. 24 is an photomicrograph showing nanocaps (half-nanoshells)comprising polystyrene nanospheres coated with silver.

FIGS. 25A-25B show various schematic embodiments of basic EIP probes.

FIGS. 26A-26E are graphical representations of fluorescence spectra ofPAH compounds.

FIG. 27 is a graph showing the XEOL of Eu doped in BaFBr matrix.

FIGS. 28A-28B provide further embodiments of schematic designs of EIPprobes.

FIGS. 29A-29B are graphical representations of various embodiments ofbasic EPEP probes.

FIGS. 30A-30C are graphical representations of various embodiments ofbasic EPEP probes.

FIGS. 31A-31B are graphical representations of various embodiments ofEPEP probes having NPs, NWs and NRs.

FIGS. 32A-32B are graphical representations of various embodiments ofEPEP probes having NPs, NWs, NRs and bioreceptors.

FIG. 33 is a graphical representation of an embodiment of EPEP probeshaving NPs and multiple NWs.

FIGS. 34A-34G show photo-active probes in which a photo-active moleculeis bound to plasmonics probes.

FIGS. 34H-34K show wavelength spectra from diamond or DLC-basedmaterials.

FIGS. 35A-35G shows plasmonics photo-active probes that have adielectric layer between the metal and the UC materials.

FIG. 36 is a plot of relative cell kill using various coatings on amixture of two phosphors compared to a control sample having nophosphor.

FIG. 37 is a representative phase diagram of a diamond like carboncoating material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention sets forth a novel method of modifying a targetstructure which mediates or is associated with a biological activity,which includes treating a condition, disorder or disease in a subject.Those cells suffering from a condition, disorder or disease are referredto herein as the target cells.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

Generally, the present invention provides a method for affecting achange in biological activity, comprising:

providing in a vicinity of or within a target structure one or morelight emitters capable of emitting different wavelengths correspondingto respective biological responses; and

activating plural biological responses in the target structure dependingon different wavelengths of light generated internally or providedinternally within the subject, wherein the different wavelengthsactivate the respective biological responses (i.e., selectiveactivation).

For example, as noted above, mechanisms involved in photoactivation of adrug such as psoralen and mechanisms involved in the formation of highlyreactive oxygen species, such as singlet oxygen may both lead to damageof tumor cells, viruses and healthy cells. For activation of the body'sown immune system, the body must identify a malignant cell or virus asthreat and create an immune response capable of lasting cytotoxiceffects directed to that threat. In the present invention, differentwavelengths of light (for example from different phosphors nearby orwithin the target cell) activate different biological response. In oneembodiment, one wavelength can be used to activate psoralen for itsattachment to a cancer cell, while another different wavelength can beused for a different purpose such as for example singlet oxygengeneration (i.e., highly reactive oxygen species), excitation of DNAstrands of the cancer cell making it more susceptible for psoraleninteraction, DNA fragmentation.

In one embodiment, one wavelength can be used to activate psoralen forits attachment to a cancer cell, while another different wavelength canbe used to stimulate psoralen such that it reacts with molecular oxygento produce active oxygen species such as superoxide and singlet oxygenthat cause lethal damage.

In various embodiments of this invention, the different wavelengths ofthe present invention can be used for regulation and control ofbiological responses having varying degrees of apoptosis (the process ofprogrammed cell death PCD) and necrosis (the premature death of cellsand living tissue typically from external factors). In necrosis, factorsexternal to the cell or tissue, such as infection, toxins, or traumathat result in the unregulated digestion of cell components. Incontrast, apoptosis is a naturally occurring programmed and targetedcause of cellular death. While apoptosis often provides beneficialeffects to the organism, necrosis is almost always detrimental and canbe fatal.

Cells that die due to necrosis do not follow the apoptotic signaltransduction pathway but rather various receptors are activated thatresult in the loss of cell membrane integrity and an uncontrolledrelease of products of cell death into the intracellular space. Thisinitiates in the surrounding tissue an inflammatory response whichprevents nearby phagocytes from locating and eliminating the dead cellsby phagocytosis. For this reason, it is often necessary to removenecrotic tissue surgically, a procedure known as debridement. Untreatednecrosis results in a build-up of decomposing dead tissue and celldebris at or near the site of the cell death. A classic example isgangrene.

In one embodiment of this invention, the different wavelengths addresseither factors which influence the progression of necrosis or itssymptoms. In one embodiment of this invention, the different wavelengthsprovides for a more “programmed” apoptosis to eliminate unhealthy cellssuch as cancer. In one embodiment of this invention, the differentwavelengths can promote interferon-beta which triggers cells to undergonecrosis, and this mechanism could kill cancer cells

Interferons (IFNs) are proteins made and released by host cells inresponse to the presence of pathogens such as viruses, bacteria,parasites or tumor cells. IFNs allow for communication between cells totrigger the protective defenses of the immune system that eradicatepathogens or tumors. IFNs belong to the large class of glycoproteinsknown as cytokines. Interferons are named after their ability to“interfere” with viral replication within host cells. IFNs have otherfunctions: they activate immune cells, such as natural killer cells andmacrophages; they increase recognition of infection or tumor cells byup-regulating antigen presentation to T lymphocytes;

In general, the different wavelengths provided to the target structureselectively turn on different biological responses. However, the presentinvention is not so limited and different wavelengths provided to thetarget structure may have a cumulative (or alternatively a synergistic)effect with regard to one biological response. Moreover, in the presentinvention, the biological response may be one which suppresses abiological reaction.

In various embodiments, the different biological responses include notonly the activation of a drug (e.g., the psoralen activation notedabove) but also the redirection of metabolic pathways, up-regulation ofcertain genes, down-regulation of certain genes, secretion of cytokines,alteration of cytokine receptor responses, or combinations thereof.

In various embodiments, a first biological response caused by light afirst wavelength can result in the bonding of a drug or pharmaceuticalagent to a critical cellular structure such as nuclear DNA, mRNA, rRNA,ribosome, mitochondrial DNA, or any other functionally importantstructures. A second biological response caused by light a secondwavelength can result in releasing metabolites which can interfere withnormal metabolic pathways. A second biological response caused by lighta second wavelength can result in altering a targeted cellular responseand/or other suitable biochemical or metabolic alterations. Increases incerebral blood flow accompanied by a significant increase in nitricoxide production have been observed in subjects treated with low levelsof 808 nm radiation.

As noted above, Bjordal et al. in “A systematic review of low levellaser therapy with location-specific doses for pain from chronic jointdisorders,” in the Australian journal of physiotherapy 49(2):107-16),the entire contents of this article are incorporated herein byreferences, describe several NIR treatments for joint disorders. In oneembodiment of this invention, one of the different wavelengths providedto the target structure can include those wavelengths described inBjordal et al. for treatment of joint disorders. In particular, in oneembodiment of this invention, one of the different wavelengths providedto the target structure can be at wavelengths of 632 nm, 820 nm, 830,nm, 904 nm, and/or 1060 nm. These wavelengths tend to reduceinflammation. In this embodiment, these wavelengths (or other similarwavelengths) can be used alone or in conjunction with other drugs suchas for example anti-inflammatories such as NSAID. In this embodiment,one of the other wavelengths provided to the target structure can be inthe ultraviolet range to induce activation of a photoactivatable drugsuch as psoralen.

Additionally, in one embodiment of this invention, one of the differentwavelengths provided to the target structure can be at wavelengths of620 nm, 680 nm, 760 nm, and 820-830 nm. Other suitable wavelengths(ranges) for photobiomodulation include 1) 613.5-623.5 nm, 2)667.5-683.7 nm, 3) 750.7-772.3 nm, 4) 812.5-846.0 nm. These wavelengths(and the wavelengths described below) are useful in the presentinvention as the different wavelengths provided to the target structureto affect photobiomodulation.

For example, light in the far-red to near-IR spectral range (as one ofthe different wavelengths provided to a target structure) can modulatevarious biological processes by activation of mitochondrial respiratorychain components, resulting in initiation of a signaling cascade thatpromotes cellular proliferation and cytoprotection. Cytochrome oxidaseis considered to be a key photoacceptor of light in the far-red tonear-IR spectral range. Cytochrome oxidase is an integral membraneprotein that contains four redox active metal centers and has a strongabsorbency in the far-red to near-IR spectral range detectable in vivoby near-IR spectroscopy. Light at 660-680 nm of irradiation (as one ofthe different wavelengths provided to a target structure) can increaseelectron transfer in cytochrome oxidase, increase mitochondrialrespiration and up-regulate cytochrome oxidase activity in neuronalcells.

Photostimulation can induce a cascade of signaling events initiated bythe initial absorption of light by cytochrome oxidase. These signalingevents may include the activation of immediate early genes,transcription factors, cytochrome oxidase subunit gene expression, and ahost of other enzymes and pathways related to increased oxidativemetabolism. Red to near-IR light stimulation (as one of the differentwavelengths provided to a target structure) of mitochondrial electrontransfer can increase the generation of reactive oxygen species. Thesemitochondrially generated reactive oxygen species may function assignaling molecules to provide communication between mitochondria andthe cysts and nucleus.

Furthermore, in this photobiomodulation embodiment, one of the otherwavelengths provided to the target structure can be in the ultravioletrange to induce activation of a photoactivatable drug such as psoralen.In this embodiment, one of the other wavelengths provided to the targetstructure can be those wavelengths noted above which tend to reduceinflammation.

In another embodiment, one of the different wavelengths provided to thetarget structure can be in the range of 400 to 700 nm to reduce thedegree of neointima formation and the incidence of restenosis (anarrowing of a blood vessel, leading to restricted blood flow)Restenosis is a common adverse event of endovascular procedures such asvascular surgery, cardiac surgery, and angioplasty. Indeed, thephenomenon of vessel restenosis, an immune response to damaged tissue,is known to be a common adverse event and is one of the leading problemswith angioplasty and stenting. Accordingly, in this embodiment, light inthe range of 400 to 700 nm wavelength range, and more specifically inthe 594-600 nm, can be provided as one of the different wavelengthsprovided to the target structure in vivo to decrease fibrointimalthickening following the arterial injury. Furthermore, in thisembodiment, one of the other wavelengths provided to the targetstructure can be in the ultraviolet range to induce activation of aphotoactivatable drug such as psoralen. In this embodiment, one of theother wavelengths provided to the target structure can be thosewavelengths noted above which tend to reduce inflammation.

In another embodiment, one of the different wavelengths provided to thetarget structure can be in the range of red to infrared for modulationof brain cell activity. In this embodiment, one of the differentwavelengths provided to the target structure can be between 630 nm and800 nm or 808 nm, in near-infrared spectrum or other wavelengthsparticularly suitable for transmission and dispersion within the graymatter and white matter of the brain. It has been shown that, within thevisible and near-infrared spectral range, white matter in both thecentral and peripheral nervous systems reflects most of the incidentpower and shows a low level of absorption and a short penetration depth.In contrast, the transmittance of the gray matter is approximately twiceas high as that of the white matter. While, in the present invention,the initiation energy (e.g., x-ray flux) can readily penetrate into therecessed areas of the brain to generate by way of energy modulationagents (down converters) near infrared light, generation of the nearinfrared light in these areas and propagation of near infrared lightthroughout the diseased cells of the brains is considered to be a highlybeneficial aspect of this invention. For example, in this embodiment,exposure of the brain cells to these wavelengths in the near infraredcan induce whole-brain metabolic and antioxidant beneficial effects suchas increases in cytochrome oxidase and superoxide dismutase activitiesand increases in cerebral blood flow. Additionally, this treatment caninclude other drugs known to have a beneficial effect on braindisorders. Furthermore, in this embodiment, one of the other wavelengthsprovided to the target structure can be in the ultraviolet range toinduce activation of a photoactivatable drug such as psoralen. In thisembodiment, one of the other wavelengths provided to the targetstructure can be those wavelengths noted above which tend to reduceinflammation. (Although the description above is directed to braindisorders, these treatments according to this invention would be usefulof the treatment of other neural conditions throughout the body.)

In another embodiment of this invention, one of the differentwavelengths provided to the target structure can be either a yellow or agreen light. As noted above, photostimulation can be used to activate alight-sensitive protein such as rhodopsin (ChR2), which can then excitethe cell expressing the opsin. It has been shown thatchannelrhodopsin-2, a monolithic protein containing a light sensor and acation channel, provides electrical stimulation of appropriate speed andmagnitude to activate neuronal spike firing. Thus, light-sensitiveproteins can be introduced into cells or live subjects via a number oftechniques including electroporation, DNA microinjection, viraldelivery, liposomal transfection and calcium-phosphate precipitation.The gene, chloride pump (NpHR), which is borrowed from a microbe calledan archaebacterium, can make neurons less active in the presence ofyellow light. By combining genes ChR2 and NpHR, neurons can be made toobey pulses of light like drivers obey a traffic signal: Blue means “go”(emit a signal), and yellow means “stop” (don't emit). Accordingly, alight-sensitive protein (for example, channelrhodopsin-2 (ChR2) andchloride pump halorhodopsin (NpHR)) can be incorporated into thelentiviral vector or other vector providing delivery of thelight-sensitive protein encoding gene into a target cell. ChR2containing a light sensor and a cation channel, provides electricalstimulation of appropriate speed and magnitude to activate neuronalspike firing, when the cells harboring Ch2R are pulsed with light. Thus,in the present invention, the photoactivation can lead to eithersuppression or activation of a biological process depending on the geneselected and the wavelength of light chosen. Furthermore, in thisembodiment, one of the other wavelengths provided to the targetstructure can be in the ultraviolet range to induce activation of aphotoactivatable drug such as psoralen. In this embodiment, one of theother wavelengths provided to the target structure can be thosewavelengths noted above which tend to reduce inflammation.

In another embodiment of this invention, one of the differentwavelengths provided to the target structure can be 632 nm light forgeneration of a light-oxygen effect (LOE), which involves activation ofor damage to biosystems by optical radiation at low optical doses bydirect photoexcitation of molecular oxygen dissolved in a biosystem sothat oxygen dissolved is converted to a singlet state, i.e., byphotogeneration of molecular singlet oxygen from 02 dissolved in cells.This process can occur in the presence or absence of a photosensitizer.Furthermore, in this embodiment, one of the other wavelengths providedto the target structure can be in the ultraviolet range to induceactivation of a photoactivatable drug such as psoralen. In thisembodiment, one of the other wavelengths provided to the targetstructure can be those wavelengths noted above which tend to reduceinflammation.

In another embodiment of this invention, one of the differentwavelengths provided to the target structure can be

While denoted above as belonging to either first or second biologicalresponses, the present invention can affect any of the biologicalresponses set forth in this specification as either a first or a secondbiological response. Furthermore, the sequence of biological responsesdoes not necessarily follow in order. For example, a first biologicalresponse could in time actually occur first or second or simultaneously.Likewise, a second biological response could actually occur in time as afirst response or a second response or simultaneously with the firstbiological response.

In one embodiment, the method applies initiation energy from at leastone source to the target structure, wherein the initiation energycontacts the target structure, generates at least one or more differentwavelengths of light, and induces a predetermined change in the targetstructure in situ.

In one embodiment, the predetermined change modifies the targetstructure or modulates the biological activity of the target structure.

In one embodiment, the method contacts the target structure with atleast one activatable pharmaceutical agent (PA) that is capable ofeffecting a predetermined change in the target structure when activatedby the one or more different wavelengths of light one of the pluralityof light emitters.

In one embodiment, the method applies an initiation energy from at leastone source to the target structure in a subject in need of treatment,wherein the initiation energy contacts the target structure and inducesa predetermined change in the target structure in situ by way of theselective activation from the plurality of light emitters at differentwavelengths. In one embodiment of the method, the predetermined changemodifies the target structure and modulates the biological activity ofthe target structure.

In one embodiment, the method contacts a target structure with at leastone activatable pharmaceutical agent (PA) that is capable of effecting apredetermined change in a target structure when activated by one of theplurality of light emitters, optionally in the presence of at least onemember selected from the group consisting of energy modulation agents,plasmonics-active agents and combinations thereof.

In one embodiment, the energy modulation agent, if present, upgrades ordowngrades the initiation energy to produce one or more of the differentwavelengths of light. In one embodiment, the plasmonics-active agent, ifpresent, enhances or modifies the light generated internally or providedinternally within the subject or applied initiation energy or both.

A further object of the present invention is to provide such methodswhich can use any suitable energy source as the initiation energy sourcein combination with plasmonics materials to activate the activatablepharmaceutical agent and thereby cause the predetermined change.

A further object of the present invention is to provide such methodsusing plasmonics in an energy cascade to activate an activatablepharmaceutical agent that then cause the predetermined change.

A further object of the present invention is to provide such methods forin situ generation of energy which causes, either directly orindirectly, the predetermined change.

A further object of the present invention is to provide a method for thetreatment of a cell proliferation disorder that permits treatment of asubject in any area of the body while being non-invasive and having highselectivity for targeted cells relative to healthy cells through the useof exciton-plasmon enhancement.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease which can use any suitableenergy source as the initiation energy source in combination withplasmon enhancement to activate the activatable pharmaceutical agent.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease using plasmon enhancementin an energy cascade to activate an activatable pharmaceutical agentthat then treats cells suffering from a condition, disorder or disease.

The condition, disorder, or disease may be mediated by abnormal cellularproliferation and the predetermined change in one embodiment canameliorate the abnormal cellular proliferation. Abnormal cellularproliferation may be higher than that of cells from a subject not havingsaid condition, disorder or disease or may be lower.

The treated condition, disorder, or disease may or may not besignificantly mediated by abnormal cellular proliferation, and thepredetermined change does not have to substantially affect cellularproliferation.

The target structure need not be present inside an organism, but may beone in vitro or ex vivo. The predetermined change may enhance theexpression of, promote the growth of, or increase the quantity of thetarget structure; or the predetermined change can enhance, inhibit orstabilize the usual biological activity of the target structure comparedto a similar untreated target structure. For example, the predeterminedchange can alter the immunological or chemical properties of the targetstructure which may be a cell, cell membrane, internal cellularstructure, polypeptide or non-polypeptide compound which can be modifiedby said predetermined change to be more or less antigenic orimmunogenic. In another embodiment, modifying the target structure canbe done without the need for a pharmaceutical agent, or a plasmonicsagent.

One object of the present invention is to modify a target structurewhich mediates or is associated with a biological activity, and in apreferred embodiment to treat a condition, disorder or disease, in asubject using photobiomodulation by way of the selective activationnoted above.

Exemplary conditions, disorders or diseases may include, but are notlimited to, cancer, autoimmune diseases, soft and bone tissue injury,chronic pain, wound healing, nerve regeneration, viral and bacterialinfections, fat deposits (liposuction), varicose veins, enlargedprostate, retinal injuries and other ocular diseases, Parkinson'sdisease, and behavioral, perceptional and cognitive disorders. Exemplaryconditions also may include nerve (brain) imaging and stimulation, adirect control of brain cell activity with light, control of cell death(apoptosis), and alteration of cell growth and division. Other exemplaryconditions, disorders or diseases may include, but are not limited tocardiac ablasion (e.g., cardiac arrhythmia and atrial fibrillation),photoangioplastic conditions (e.g., de novo atherosclerosis,restinosis), intimal hyperplasia, arteriovenous fistula, maculardegeneration, psoriasis, acne, hopecia areata, portwine spots, hairremoval, rheumatoid and inflammatory arthrisis, joint conditions, lymphnode conditions, and cognitive and behavioral conditions.

In one embodiment, a method in accordance with the present inventionutilizes the principle of energy transfer to and among molecular agentsto control delivery and activation of cellular changes by irradiationsuch that delivery of the desired effect is more intensified, precise,and effective than the conventional techniques. At least one energymodulation agent can be administered to the subject which adsorbs,intensifies or modifies said initiation energy into an energy thateffects a predetermined cellular change in said target structure. Theenergy modulation agent may be located around, on, or in said targetstructure. Further, the energy modulation agent can transform a photonicinitiation energy into a photonic energy that effects a predeterminedchange in said target structure. In one embodiment, the energymodulation agent decreases the wavelength of the photonic initiationenergy (down convert). In another embodiment, the energy modulationagent can increase the wavelength of the photonic initiation energy (upconvert). In a different embodiment the modulation agent is one or moremembers selected from a biocompatible fluorescing metal nanoparticle,fluorescing metal oxide nanoparticle, fluorescing dye molecule, goldnanoparticle, silver nanoparticle, gold-coated silver nanoparticle, awater soluble quantum dot encapsulated by polyamidoamine dendrimers, aluciferase, a biocompatible phosphorescent molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelateexhibiting intense luminescence.

In one aspect of the invention, a downconverting energy modulation agentcan comprise inorganic particulates selected from the group consistingof: metal oxides; metal sulfides; doped metal oxides; and mixed metalchalcogenides. In one aspect of the invention, the downconvertingmaterial can comprise at least one of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄, YAG,YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, Na-dopedYbF₃, ZnS; ZnSe; MgS; CaS and alkali lead silicate includingcompositions of SiO₂, B₂O₃, Na₂O, K₂O, PbO, MgO, or Ag, and combinationsor alloys or layers thereof. In one aspect of the invention, thedownconverting material can include a dopant including at least one ofEr, Eu, Yb, Tm, Nd, Mn Tb, Ce, Y, U, Pr, La, Gd and other rare-earthspecies or a combination thereof. The dopant can be included at aconcentration of 0.01%-50% by mol concentration.

In one aspect of the invention, the downconverting energy modulationagent can comprise materials such as ZnSeS:Cu, Ag, Ce, Tb; CaS: Ce,Sm;La₂O₂S:Tb; Y₂O₂S:Tb; Gd₂O₂S:Pr, Ce, F; LaPO₄. In other aspects of theinvention, the downconverting material can comprise phosphors such asZnS:Ag and ZnS:Cu, Pb. In other aspects of the invention, thedownconverting material can be alloys of the ZnSeS family doped withother metals. For example, suitable materials include ZnSe_(x)S_(y):Cu,Ag, Ce, Tb, where the following x, y values and intermediate values areacceptable: x:y; respectively 0:1; 0.1:0.9; 0.2:0.8; 0.3:0.7; 0.4:0.6;0.5:0.5;

0.6:0.4; 0.7:0.3; 0.8:0.2; 0.9:0.1; and 1.0:0.0.

In other aspects of the invention, the downconverting energy modulationagent can be materials such as sodium yttrium fluoride (NaYF₄),lanthanum fluoride (LaF₃), lanthanum oxysulfide (La₂O₂S), yttriumoxysulfide (Y₂O₂S), yttrium fluoride (YF₃), yttrium gallate, yttriumaluminum garnet (YAG), gadolinium fluoride (GdF₃), barium yttriumfluoride (BaYF₅, BaY₂F₈), gadolinium oxysulfide (Gd₂O₂S), calciumtungstate (CaWO₄), yttrium oxide:terbium (Yt₂O₃Tb), gadoliniumoxysulphide:europium (Gd₂O₂S:Eu), lanthanum oxysulphide:europium(La₂O₂S:Eu), and gadolinium oxysulphide:promethium, cerium, fluorine(Gd₂O₂S:Pr,Ce,F), YPO₄:Nd, LaPO₄:Pr, (Ca,Mg)SO₄:Pb, YBO₃:Pr, Y₂SiO₅:Pr,Y₂Si₂O₇:Pr, SrLi₂SiO₄:Pr,Na, and CaLi₂SiO₄:Pr.

In other aspects of the invention, the downconverting energy modulationagent can be near-infrared (NIR) downconversion (DC) phosphors such asKSrPO₄:Eu²⁺, Pr³⁺, or NaGdF₄:Eu or Zn₂SiO₄:Tb³⁺,Yb³⁺ or β-NaGdF₄co-doped with Ce³⁺ and Tb³⁺ ions or Gd₂O₂S:Tm or BaYF₅:Eu³⁺ or otherdown converters which emit NIR from visible or UV light exposure (as ina cascade from x-ray to UV to NIR) or which emit NIR directly afterx-ray or e-beam exposure.

In one aspect of the invention, a up converting energy modulation agentcan be at least one of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄, YAG, YAP, Nd₂O₃,LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, Na-doped YbF₃, or SiO₂or alloys or layers thereof.

In one aspect of the invention, the energy modulation agents can be usedsingly or in combination with other down converting or up convertingmaterials.

In one embodiment, the initiation energy can be provided by way ofcatheters inserted into the subject. The catheters can includeprescribed energy modulation agents (e.g., up converting or downconverting materials noted herein) at a distal end of the catheter whichemit the aforementioned light for selective activation into the subject.

Below is a list of X-ray phosphors which can be used in the presentinvention along with their corresponding peak emission values.

TABLE 1 Emission Spectrum X-ray Absorption Microstructure Peak EmissionEmiss Eff K-edge Specific Crystal # Phosphor (nm) (%) Eff (Z) (keV)Gravity Structure Hygroscopic 1 BaFCl:Eu²⁺ 380 13 49.3 37.38 4.7Tetragonal N 2 BaSO₄−:Eu²⁺ 390 6 45.5 37.38 4.5 Rhombic N 3 LaOBr:Tm³⁺360, 460 14 49.3 38.92 6.3 Tetragonal N 4 YTaO₄ 337 59.8 67.42 7.5Monolithic N 5 YTaO₄:Nb (*) 410 11 59.8 67.42 7.5 Monolithic N 6 CaWO₄420 5 61.8 69.48 6.1 Tetragonal N 7 LaOBr:Tb³⁺ 420 20 49.3 38.92 6.3Tetragonal N 8 Y₂O₂S:Tb³⁺ 420 18 34.9 17.04 4.9 Hexgonal N 9 ZnS:Ag 45017 26.7  9.66 3.9 Hexgonal N 10 (Zn,Cd)S:Ag 530 19 38.4 9.66/26.7 4.8Hexgonal N 11 Gd₂O₂S:Tb³⁺ 545 13 59.5 50.22 7.3 Hexgonal N 12La₂O₂S:Tb³⁺ 545 12.5 52.6 38.92 6.5 Hexgonal NVarious plastic scintillators, plastic scintillator fibers and relatedmaterials are made of polyvinyltoluene or styrene and fluors. Thesematerials could be used in the present invention especially ifencapsulated or otherwise chemically isolated from the target structureso not as to be dissolved or otherwise deteriorated by the fluids of thetarget structure. These and other formulations are commerciallyavailable, such as from Saint Gobain Crystals, as BC-414, BC-420,BC-422, or BCF-10.

TABLE 2 Product Peak Emission Phosphor Reference (nm) Organic BC-414 392Organic BC-420 391 Organic BC-422 370Other polymers are able to emit in the visible range and these include:

TABLE 3 # of Phosphor Product Peak Emission Photons Per (Fiber Forms)Reference (nm) MeV Organic BCF-10 432 8000 Organic BC-420 435 8000Organic BC-422 492 8000

Table 4 shows a wide variety of energy modulation agents which can beused in this invention.

TABLE 4 Emission Spectrum X-Ray Peak Emiss Absoprtion Emmission Eff EffK-edge Specific Crystal Phosphor Color (nm) (%) (Z) (keV) GravityStructure Hygroscopic Zn3(PO4)2: Tl+ 310 N BaF2 310 Slightly CsI 315 NCa3(PO4)2: Tl+ 330 N YTaO4 337 59.8 67.42 7.5 Monolithic N CsI: Na 338 YBaSi2O5: Pb2+ 350 N Borosilicate 350 N LaCl3(Ce) 350 Y SrB4O7F: Eu2+ 360N RbBr: Tl+ 360 ? (Ba, Sr, Mg)3Si2O7: Pb2+ 370 N YAlO3: Ce3+ 370 NBC-422 370 Organic ? BaFCl: Eu2+ 380 13 49.3 37.38 4.7 Tetragonal NBaSO4—: Eu2+ 390 6 45.5 37.38 4.5 Rhombic N BaFBr: Eu2+ 390 ? BC-420 391Organic ? BC-414 392 Organic ? SrMgP2O7: Eu2+ 394 N BaBr2: Eu2+ 400 N(Sr, Ba)Al2Si2O8: Eu2+ 400 N YTaO4: Nb (*) 410 11 59.8 67.42 7.5Monolithic N Y2SiO5: Ce3+ 410 N CaWO4 420 5 61.8 69.48 6.1 Tetragonal NLaOBr: Tb3+ 420 20 49.3 38.92 6.3 Tetragonal N Y2O2S: Tb3+ 420 18 34.917.04 4.9 Hexgonal N Lu2SiO5: Ce3+ 420 N Lu1.8 Y0.2SiO5: Ce 420 N ZnS:Ag 450 17 26.7 9.66 3.9 Hexgonal N CdWO4 475 Slightly Bi4Ge3O12 (BGO)480 N (Zn, Cd)S: Ag 530 19 38.4 9.66/26.7 4.8 Hexgonal N Gd2O2S: Tb3+545 13 59.5 50.22 7.3 Hexgonal N La2O2S: Tb3+ 545 12.5 52.6 38.92 6.5Hexgonal N Y3Al5O12 (Ce) 550 N LaOBr: Tm3+ 360,460 14 49.3 38.92 6.3Tetragonal N CaF2(Eu) 435/300 N

By selection of one or more of the phosphors noted above (or othersknown in the art), the present invention permits one to provide in avicinity of or within a target structure one or more light emitterscapable of emitting different wavelengths corresponding to respectivebiological responses, and permits the activation of one or morebiological responses in the target structure depending on at least oneor more different wavelengths of light generated internally or providedinternally within the subject, wherein the different wavelengthsactivate the respective biological responses (i.e., selectiveactivation).

In one embodiment, the present invention provides methods utilizing theprinciple of energy transfer to and among molecular agents to controldelivery and activation of pharmaceutically active agents such thatdelivery of the desired pharmacological effect is focused and precise.

In one embodiment, the initiation energy source is applied directly orindirectly (via a modulation agent) to the activatable pharmaceuticalagent, preferably in proximity to the target cells.

Within the context of the present invention, the phrase “appliedindirectly” (or variants of this phrase, such as “applying indirectly”,“indirectly applies”, “indirectly applied”, “indirectly applying”,etc.), when referring to the application of the initiation energy, meansthe penetration by the initiation energy into the subject beneath thesurface of the subject and to the modulation agent and/or activatablepharmaceutical agent within a subject. In one embodiment, the initiationenergy interacts with a previously administered energy modulation agentwhich then activates the predetermined cellular changes. In anotherembodiment, the initiation energy interacts with a previouslyadministered energy modulation agent which then activates theactivatable pharmaceutical agent. In another embodiment, the initiationenergy itself activates the activatable pharmaceutical agent. In eitherembodiment, the initiation energy source cannot be within line-of-sightof the modulation agent and/or the activatable pharmaceutical agent. By“cannot be within line-of-sight” is meant that if a hypotheticalobserver were located at the location of the modulation agent or theactivatable pharmaceutical agent, that observer would be unable to seethe source of the initiation energy.

Although not intending to be bound by any particular theory or beotherwise limited in any way, the following theoretical discussion ofscientific principles and definitions are provided to help the readergain an understanding and appreciation of the present invention.

As used herein, the term “subject” is not intended to be limited tohumans, but may also include animals, plants, or any suitable biologicalorganism.

As used herein, the phrase “a disease or condition” refers to acondition, disorder or disease that may include, but are not limited to,cancer, soft and bone tissue injury, chronic pain, wound healing, nerveregeneration, viral and bacterial infections, fat deposits(liposuction), varicose veins, enlarged prostate, retinal injuries andother ocular diseases, Parkinson's disease, and behavioral, perceptionaland cognitive disorders. Exemplary conditions also may include nerve(brain) imaging and stimulation, a direct control of brain cell activitywith light, control of cell death (apoptosis), and alteration of cellgrowth and division. Yet other exemplary a condition, disorder ordisease may include, but are not limited to, cardiac ablasion (e.g.,cardiac arrhythmia and atrial fibrillation), photoangioplasticconditions (e.g., de novo atherosclerosis, restinosis), intimalhyperplasia, arteriovenous fistula, macular degeneration, psoriasis,acne, hopecia areata, portwine spots, hair removal, rheumatoid andinflammatory arthritis, joint conditions, and lymph node conditions.

As used herein, the term “target structure” refers to an eukaryoticcell, prokaryotic cell, a subcellular structure, such as a cellmembrane, a nuclear membrane, cell nucleus, nucleic acid, mitochondria,ribosome, or other cellular organelle or component, an extracellularstructure, virus or prion, and combinations thereof.

The nature of the predetermined cellular change will depend on thedesired pharmaceutical outcome. Exemplary cellular changes may include,but are not limited to, apoptosis, necrosis, up-regulation of certaingenes, down-regulation of certain genes, secretion of cytokines,alteration of cytokine receptor responses, regulation of cytochrome coxidase and flavoproteins, activation of mitochondria, stimulationantioxidant protective pathway, modulation of cell growth and division,alteration of firing pattern of nerves, alteration of redox properties,generation of reactive oxygen species, modulation of the activity,quantity, or number of intracellular components in a cell, modulation ofthe activity, quantity, or number of extracellular components producedby, excreted by, or associated with a cell, or a combination thereof.Predetermined cellular changes may or may not result in destruction orinactivation of the target structure.

As used herein, an “energy modulation agent” refers to an agent that iscapable of receiving an energy input from a source and then re-emittinga different energy to a receiving target. Energy transfer amongmolecules may occur in a number of ways. The form of energy may beelectronic, thermal, electromagnetic, kinetic, or chemical in nature.Energy may be transferred from one molecule to another (intermoleculartransfer) or from one part of a molecule to another part of the samemolecule (intramolecular transfer). For example, a modulation agent mayreceive electromagnetic energy and re-emit the energy in the form ofthermal energy. In preferred embodiments, the energy modulation agentreceives higher energy (e.g. x-ray) and re-emits in lower energy (e.g.UV-A). Some modulation agents may have a very short energy retentiontime (on the order of fs, e.g. fluorescent molecules) whereas others mayhave a very long half-life (on the order of minutes to hours, e.g.luminescent or phosphorescent molecules). Suitable energy modulationagents include, but are not limited to, a biocompatible fluorescingmetal nanoparticle, fluorescing dye molecule, gold nanoparticle, a watersoluble quantum dot encapsulated by polyamidoamine dendrimers, aluciferase, a biocompatible phosphorescent molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelatecapable of intense luminescence. Various exemplary uses of these aredescribed below in preferred embodiments.

The modulation agents may further be coupled to a carrier for cellulartargeting purposes. For example, a biocompatible molecule, such as afluorescing metal nanoparticle or fluorescing dye molecule that emits inthe UV-A band, may be selected as the energy modulation agent.

The energy modulation agent (or agents) may be preferably directed tothe desired site (e.g. a tumor) by systemic administration to a subject.For example, a UV-A emitting energy modulation agent (or agents) may beconcentrated in the tumor site by physical insertion or by conjugatingthe UV-A emitting energy modulation agent with a tumor specific carrier,such as a lipid, chitin or chitin-derivative, a chelate or otherfunctionalized carrier that is capable of concentrating the UV-Aemitting source in a specific target tumor.

In one embodiment, the energy modulation agent can be used alone or as aseries of two or more energy modulation agents wherein the energymodulation agents provide an energy cascade. Thus, the first energymodulation agent in the cascade will absorb the activation energy,convert it to a different energy which is then absorbed by the secondenergy modulation in the cascade, and so forth until the end of thecascade is reached with the final energy modulation agent in the cascadeemitting the energy necessary to activate the activatable pharmaceuticalagent.

Exemplary energy modulation agents may include, but are not limited to,at least one energy modulation agent selected from the group consistingof a biocompatible fluorescing metal nanoparticle, fluorescing metaloxide nanoparticle, fluorescing dye molecule, gold nanoparticle, silvernanoparticle, gold-coated silver nanoparticle, a water soluble quantumdot encapsulated by polyamidoamine dendrimers, a luciferase, abiocompatible phosphorescent molecule, a combined electromagnetic energyharvester molecule, and a lanthanide chelate exhibiting intenseluminescence.

As used herein, an “activatable pharmaceutical agent” is an agent thatnormally exists in an inactive state in the absence of an activationsignal. When the agent is activated by a matching activation signalunder activating conditions, it is capable of effecting the desiredpharmacological effect on a target cell (i.e. preferably a predeterminedcellular change).

Signals that may be used to activate a corresponding agent may include,but are not limited to, photons of specific wavelengths (e.g. x-rays, orvisible light), electromagnetic energy (e.g. radio or microwave),thermal energy, acoustic energy, or any combination thereof.

Activation of the agent (or agents) may be as simple as delivering thesignal to the agent or may further premise on a set of activationconditions. For example, in the former case, an activatablepharmaceutical agent, such as a photosensitizer, may be activated byUV-A radiation. Once activated, the agent (or agents) in its (their)active-state may then directly proceed to effect a cellular change.

Where activation may further premise upon other conditions, meredelivery of the activation signal may not be sufficient to bring aboutthe desired cellular change. For example, a photoactive compound thatachieves its pharmaceutical effect by binding to certain cellularstructure in its active state may require physical proximity to thetarget cellular structure when the activation signal is delivered. Forsuch activatable agents, delivery of the activation signal undernon-activating conditions will not result in the desired pharmacologiceffect. Some examples of activating conditions may include, but are notlimited to, temperature, pH, location, state of the cell, presence orabsence of co-factors.

Selection of an activatable pharmaceutical agent greatly depends on anumber of factors such as the desired cellular change, the desired formof activation, as well as the physical and biochemical constraints thatmay apply. Exemplary activatable pharmaceutical agents may include, butare not limited to, agents that may be activated by photonic energy,electromagnetic energy, acoustic energy, chemical or enzymaticreactions, thermal energy, or any other suitable activation mechanisms.

When activated, the activatable pharmaceutical agent may effect cellularchanges that include, but are not limited to, apoptosis, redirection ofmetabolic pathways, up-regulation of certain genes, down-regulation ofcertain genes, secretion of cytokines, alteration of cytokine receptorresponses, or combinations thereof.

The mechanisms by which an activatable pharmaceutical agent may achieveits desired effect are not particularly limited. Such mechanisms mayinclude direct action on a predetermined target as well as indirectactions via alterations to the biochemical pathways. A preferred directaction mechanism is by binding the agent to a critical cellularstructure such as nuclear DNA, mRNA, rRNA, ribosome, mitochondrial DNA,or any other functionally important structures. Indirect mechanisms mayinclude releasing metabolites upon activation to interfere with normalmetabolic pathways, releasing chemical signals (e.g. agonists orantagonists) upon activation to alter the targeted cellular response,and other suitable biochemical or metabolic alterations.

The treatment of the present invention can be by the methods describedin U.S. application Ser. No. 11/935,655, filed Nov. 6, 2007(incorporated by reference above), or by a modified version of aconventional treatment such as PDT, but using a plasmonics-active agentto enhance the treatment by modifying or enhancing the applied energyor, in the case of using an energy modulation agent, modifying eitherthe applied energy, the emitted energy from the energy modulation agent,or both.

In one preferred embodiment, the activatable pharmaceutical agent iscapable of chemically binding to the DNA or mitochondria at atherapeutically effective amount. In this embodiment, the activatablepharmaceutical agent, preferably a photoactivatable agent, is exposed insitu to an activating energy emitted from an energy modulation agent (oragents), which had received energy from an initiation energy source.

Suitable activatable agents include, but are not limited to, photoactiveagents, sono-active agents, thermo-active agents, andradio/microwave-active agents. An activatable agent may be a smallmolecule; a biological molecule such as a protein, a nucleic acid orlipid; a supramolecular assembly; a nanoparticle; or any other molecularentity having a pharmaceutical activity once activated.

The activatable agent may be derived from a natural or synthetic origin.Any such molecular entity that may be activated by a suitable activationsignal source to effect a predetermined cellular change may beadvantageously employed in the present invention.

Suitable photoactive agents include, but are not limited to: psoralensand psoralen derivatives, pyrene cholesteryloleate, acridine, porphyrin,fluorescein, rhodamine, 16-diazorcortisone, ethidium, transition metalcomplexes of bleomycin, transition metal complexes of deglycobleomycin,organoplatinum complexes, alloxazines such as 7,8-dimethyl-10-ribitylisoalloxazine (riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin),7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide(flavine adenine dinucleotide [FAD]), alloxazine mononucleotide (alsoknown as flavine mononucleotide [FMN] and riboflavine-5-phosphate),vitamin Ks, vitamin L, their metabolites and precursors, andnapththoquinones, naphthalenes, naphthols and their derivatives havingplanar molecular conformations, porphyrins, dyes such as neutral red,methylene blue, acridine, toluidines, flavine (acriflavinehydrochloride) and phenothiazine derivatives, coumarins, quinolones,quinones, and anthroquinones, aluminum (111) phthalocyaninetetrasulfonate, hematoporphyrin, and phthalocyanine, and compounds whichpreferentially adsorb to nucleic acids with little or no effect onproteins. The term “alloxazine” includes isoalloxazines.

Endogenously-based derivatives include synthetically derived analogs andhomologs of endogenous photoactivated molecules, which may have or lacklower (1 to 5 carbons) alkyl or halogen substituents of thephotosensitizers from which they are derived, and which preserve thefunction and substantial non-toxicity. Endogenous molecules areinherently non-toxic and may not yield toxic photoproducts afterphotoradiation.

Table 5 lists some photoactivatable molecules capable of beingphotoactivated to induce an auto vaccine effect.

TABLE 5 k_(s) of Com- A_(ex) donor k_(SSET)(s⁻¹) R₀ R R_(model)(A)k_(TTET) pound (nm) E_(SSET) (s⁻¹) k_(SSET) (s⁻¹) (Average) (A) (A)(Average) E_(TTET) (s⁻¹) 1B 224 96.3 9.5 × 10⁵ 2.44 × 10⁸ 1.87 × 10³14.7 9 9.5 266 95  1.8 × 10⁸ 2.5   5 × 10² 280 94 1.36 × 10⁸ 1A 224 809.5 × 10⁵  3.8 × 10⁷ 3.67 × 10⁷ 14.7 11.8 14.1 266 79  3.6 × 10⁷ 2 3.6 ×10² 280 79  3.6 × 10⁷ 2B 224 77 9.5 × 10⁵  3.1 × 10⁷  3.9 × 10⁷ 14.711.9 6.5 266 81  3.9 × 10⁷ 32 9.4 × 10³ 280 83  4.7 × 10⁷ 2A 224 69 9.5× 10⁵  2.1 × 10⁷   3 × 10⁷ 14.7 12.2 8.1 74.3 5.7 × 10⁴ 266 80  3.7 ×10⁷ 280 77  3.2 × 10⁷

Table 6 lists some additional endogenous photoactivatable molecules.

TABLE 6 Excitation Max. Emission Max. Endogenous Fluorophores (nm) (nm)Amino acids: Tryptophan 280 350 Tyrosine 275 300 Phenylalanine 260 280Structural Proteins: Collagen 325, 360 400, 405 Elastin 290, 325 340,400 Enzymes and Coenzymes: flavin adenine dinucleotide 450 535 reducednicotinamide dinucelotide 290, 351 440, 400 reduced nicotinamidedinucelotide 336 464 phosphate Vitamins Vitamins A 327 510 Vitamins K335 480 Vitamins D 390 480 Vitamins B₆ compounds: Pyridoxine 332, 340400 Pyridoxamine 335 400 Pyridoxal 330 385 Pyridoxic acid 315 425Pyridoxal phosphate  5′-330 400 Vitamin B₁₂ 275 305 Lipids:Phospholipids 436 540, 560 Lipofuscin 340-395 540, 430-460 Ceroid340-395 430-460, 540 Porphyrine 400-450 630, 690

FIG. 1 provides an exemplary electromagnetic spectrum in meters (1 nmequals meters).

Although the activatable pharmaceutical agent and the energy modulationagent can be distinct and separate, it will be understood that the twoagents need not be independent and separate entities. In fact, the twoagents may be associated with each other via a number of differentconfigurations. Where the two agents are independent and separatelymovable from each other, they generally interact with each other viadiffusion and chance encounters within a common surrounding medium.Where the activatable pharmaceutical agent and the energy modulationagent are not separate, they may be combined into one single entity.

The initiation energy source can be any energy source capable ofproviding energy at a level sufficient to cause cellular changesdirectly or via a modulation agent which transfer the initiation energyto energy capable of causing the predetermined cellular changes. Also,the initiation energy source can be any energy source capable ofproviding energy at a level sufficient activate the activatable agentdirectly, or to provide the energy to a modulation agent with the inputneeded to emit the activation energy for the activatable agent (indirectactivation). Preferable initiation energy sources include, but are notlimited to, UV-A lamps or fiber optic lines, a light needle, anendoscope, and a linear accelerator that generates x-ray, gamma-ray, orelectron beams.

In a preferred embodiment the initiation energy is capable ofpenetrating completely through the subject. Within the context of thepresent invention, the phrase “capable of penetrating completely throughthe subject” is used to refer to energy that can penetrate to any depthwithin the subject to activate the activatable pharmaceutical agent. Itis not required that the any of the energy applied actually passcompletely through the subject, merely that it be capable of doing so inorder to permit penetration to any desired depth to activate theactivatable pharmaceutical agent. Exemplary initiation energy sourcesthat are capable of penetrating completely through the subject include,but are not limited to, UV light, visible light, IR radiation, x-rays,gamma rays, electron beams, microwaves and radio waves. In oneembodiment, the initiation energy can penetrate completely through thesubject and can be applied from a single source or more than one source.

In another embodiment, the initiation energy source 1 may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. One example of such is the SmartBeam™ IMRT (intensitymodulated radiation therapy) method from Varian medical methods (VarianMedical Methods, Inc., Palo Alto, Calif.). X-ray machines that producefrom 10 to 150 keV X-rays are readily available in the marketplace. Forinstance, the General Electric Definium series or the Siemens MULTIXseries are but two examples of typical X-ray machines designed for themedical industry, which could be used in the present invention.

In one embodiment, the initiation energy may also be UV radiation,visible light, infrared radiation (IR), x-rays, gamma rays, an electronbeam, microwaves or radio waves. Energy modulation agents (e.g., upconverting or down converting agents) inside the subject emit theaforementioned light for selective activation into the subject.

An additional embodiment of the present invention is to provide a methodfor treatment of a condition, disease or disorder by the in-situgeneration of energy in a subject in need thereof, where the energygenerated can be used directly to effect a change thereby treating thecondition, disease or disorder, or the energy can be used to activate anactivatable pharmaceutical agent, which upon activation effects a changethereby treating the condition, disease or disorder. The energy can begenerated in-situ by any desired method, including, but not limited toconversion of an energy applied to the subject externally, which isconverted in-situ to a different energy (of lower or higher energy thanthat applied), through the use of one or more energy modulation agentsproducing at least two different wavelengths of light, each wavelengthof light associated with a different biological response. For example,light of first wavelength photactivates a pharmaceutical agent, andlight of second wavelength heats the local treatment area.

A further embodiment of the present invention combines the treatment ofa condition, disease or disorder with the generation of heat in theaffected target structure in order to enhance the effect of thetreatment. For example, in the treatment of a cell proliferationdisorder using a photoactivatable pharmaceutical agent (such as apsoralen or derivative thereof), one can activate the photoactivatablepharmaceutical agent by applying an initiation energy which, directly orindirectly, activates the pharmaceutical agent or agents by way ofexposure of the pharmaceutical agent or agents to at least two differentwavelengths of light, each wavelength of light associated with adifferent biological response. For example, light of first wavelengthphotactivates a psoralen or derivative thereof, and light of secondwavelength heats the local treatment area.

As noted elsewhere in the present application, this initiation energycan be of any type, so long as it can be converted to an energy suitablefor activating the pharmaceutical compound. In addition to applying thisinitiation energy, in this embodiment of the present invention, anenergy is applied that causes heating of the target structure. In thecase of a cell proliferation disorder such as cancer, the heating wouldincrease the proliferation rate of the cancer cells. While this may seemcounterintuitive at first, when the cell proliferation disorder is beingtreated using a DNA intercalation agent, such as psoralen or aderivative thereof, this increase in cell proliferation can actuallyassist the psoralen in causing apoptosis. In particular, when psoralenbecomes intercalated into DNA, apoptosis occurs when the cell goesthrough its next division cycle. By increasing the rate at which thecells divide, one can use the present invention methods to enhance theonset of apoptosis.

Additional sources of heat can be utilized. Heat can be generated usingthe application of microwaves or NIR energy to the target structure orby the use of use of nanoparticles of metal or having metal shells. Inthe nanoparticles embodiment, as is done in tumor thermotherapy,magnetic metal nanoparticles can be targeted to cancer cells usingconventional techniques, then used to generate heat by application of amagnetic field to the subject under controlled conditions. (DeNardo S J,DeNardo G L, Natarajan A et al.: Thermal dosimetry predictive ofefficacy of 111In-ChL6 NPAMF-induced thermoablative therapy for humanbreast cancer in mice. J. Nucl. Med. 48(3), 437-444 (2007).)

Alternatively, one can generate heat through the application of NIR tonanoparticles having metal shells which is converted into thermalenergy. (Hirsch L R, Stafford R J, Bankson J et al.: Nanoshell-mediatednear-infrared thermal therapy of tumors under magnetic resonanceguidance. Proc. Natl Acad. Sci. USA 100(23), 13549-13554 (2003)).

Photoactivatable agents may be in general stimulated by an energysource, such as UV or visible or infrared irradiation from the energymodulation agents at different wavelengths, resonance energy transfer,exciton migration, electron injection, or chemical reaction, to anactivated energy state that is capable of effecting the predeterminedcellular change desired. In a preferred embodiment, the photoactivatableagent, upon activation, binds to DNA or RNA or other structures in acell. The activated energy state of the agent is capable of causingdamage to cells, inducing apoptosis.

One preferred method of treating a condition, disorder or diseasemediated by a target structure in a subject comprises:

-   -   (1) administering to the subject at least one activatable        pharmaceutical agent that is capable of effecting a        predetermined change to the target structure when activated; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the applied initiation energy activates the activatable        agent in situ by different wavelengths of UV or visible or        infrared irradiation emitted from at least one energy modulation        agent in a vicinity of or within the target structure,    -   thus causing the predetermined change to the target structure to        occur, wherein said predetermined change treats the condition,        disorder, or disease.

Another preferred method for treating a condition, disorder or diseasemediated by a target structure in a subject, comprises:

-   -   (1) administering to the subject at least one activatable        pharmaceutical agent that is capable of activation by a multi        photon absorption event and of effecting a predetermined change        in said target when activated by different wavelengths of UV or        visible or infrared irradiation emitted from at least one energy        modulation agent in a vicinity of or within the target        structure; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the applied initiation energy activates the activatable        agent by the multi photon absorption event in situ,    -   thus causing the predetermined change to occur, wherein said        predetermined change treats the condition, disorder, or disease.

The concept of multi-photon excitation is based on the idea that two ormore photons of low energy can excite a fluorophore in a quantum event,resulting in the emission of a fluorescence photon, typically at ahigher energy than the two or more excitatory photons.

Commonly used fluorophores have excitation spectra in the 400-500 nmrange, whereas the laser used to excite the fluorophores lies in the˜700-1000 nm (infrared) range. If the fluorophore absorbs two infraredphotons simultaneously, it will absorb enough energy to be raised intothe excited state. The fluorophore will then emit a single photon with awavelength that depends on the type of fluorophore used (typically inthe visible spectrum). Because two photons need to be absorbed to excitea fluorophore, the probability of emission is related to the intensitysquared of the excitation beam. Therefore, a higher amount of two-photonfluorescence is generated where the laser beam is tightly focused thanwhere it is more diffuse. Effectively, fluorescence is observed in anyappreciable amount in the focal volume, resulting in a high degree ofrejection of out-of-focus objects.

Accordingly, another method for treating a condition, disease, ordisorder mediated by a target structure in a subject, comprises:

-   -   (1) administering to the subject at least one energy modulation        agent and at least one activatable pharmaceutical agent that is        capable of activation by multi photon absorption and of        effecting a predetermined cellular change when activated by        different wavelengths of UV or visible or infrared irradiation        emitted from the at least one energy modulation agent in a        vicinity of or within the target structure; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the energy modulation agent upgrades the applied        initiation energy to an energy, which then activates the        activatable agent by a multi photon absorption event in situ,        -   thus causing the predetermined cellular change to occur,            wherein said predetermined cellular change treats the            condition, disease or disorder.

In one embodiment, the energy upgrades are obtained via 2, 3, 4, or 5simultaneous photon absorptions.

Yet another preferred method for treating a condition, diseases, ordisorder mediated by a target structure in a subject, comprises:

-   -   (1) administering to the subject at least one energy modulation        agent and at least one activatable pharmaceutical agent that is        capable effecting a predetermined cellular change when activated        by different wavelengths of UV or visible or infrared        irradiation emitted from the at least one energy modulation        agent in a vicinity of or within the target structure; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the energy modulation agent upgrades the applied        initiation energy to an energy, which then activates the        activatable agent in situ,        -   thus causing the predetermined cellular change to occur,            wherein said predetermined cellular change treats the            condition, disease or disorder.

In yet another aspect, the radiative energy may be of a higher energythan the excitation energy of the photoactive agent. In this aspect, thephotoactive agent may be activated via an “energy downgrade” mechanism.In one scenario, via the multi-photon mechanism, two lower energyphotons having energy x may be absorbed by an agent to excite the agentfrom ground state E0 to a higher energy state E2. The agent may thenrelax down to an intermediate energy state E1 by emitting a photonhaving an energy y that is equal to the energy gap between E2 and E1,where y is less than x. Other mechanisms of energy downgrade may bemediated by energy transformation agents such as quantum dots,nanotubes, or other agents having suitable photo-radiation properties.Thus, yet another preferred method for treating a condition, disease, ordisorder mediated by a target structure in a subject, comprises:

-   -   (1) administering to the subject at least one energy modulation        agent and at least one activatable pharmaceutical agent that is        capable of activation by multi photon absorption and of        effecting a predetermined cellular change when activated by        different wavelengths of UV or visible or infrared irradiation        emitted from the at least one energy modulation agent in a        vicinity of or within the target structure; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the energy modulation agent downgrades the applied        initiation energy to an energy, which then activates the        activatable agent by a multi photon absorption event in situ,        -   thus causing the predetermined cellular change to occur,            wherein said predetermined cellular change treats the            condition, disease, or disorder.

Thus, yet another preferred method for treating a condition, disease, ordisorder mediated by a target structure in a subject, comprises:

-   -   (1) administering to the subject at least one energy modulation        agent and at least one activatable pharmaceutical agent that is        capable of effecting a predetermined cellular change when        activated by different wavelengths of UV or visible or infrared        irradiation emitted from the at least one energy modulation        agent in a vicinity of or within the target structure; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the energy modulation agent downgrades the applied        initiation energy to an energy, which then activates the        activatable agent in situ,        -   thus causing the predetermined cellular change to occur,            wherein said predetermined cellular change treats the            condition, disorder or disease.

In a further preferred embodiment, the present invention provides amethod for treating a condition, disorder or disease mediated by atarget structure in a subject, comprising:

(1) administering to the subject an activatable pharmaceutical agentthat is capable of effecting a predetermined change in said targetstructure when activated by different wavelengths of UV or visible orinfrared irradiation emitted from at least one energy modulation agentin a vicinity of or within the target structure; and

(2) applying an initiation energy from an initiation energy source tothe subject,

wherein the initiation energy applied and activatable pharmaceuticalagent upon activation produce insufficient singlet oxygen in the subjectto produce cell lysis, and wherein the initiation energy activates theactivatable pharmaceutical agent in situ,

-   -   thus causing the predetermined change to occur via said target        structure, wherein said predetermined change targets the        condition, disorder or disease.

In a further preferred embodiment, the present invention provides amethod for treating a condition, disorder or disease mediated by atarget structure in a subject, comprising:

(1) administering to the subject an activatable pharmaceutical agentthat is capable of effecting a predetermined change in said targetstructure when activated by different wavelengths of UV or visible orinfrared irradiation emitted from at least one energy modulation agentin a vicinity of or within the target structure; and

(2) applying an initiation energy from an initiation energy source tothe subject,

wherein the initiation energy applied and activatable pharmaceuticalagent upon activation produce a controlled amount of singlet oxygen inthe subject to produce cell lysis, and wherein the initiation energyactivates the activatable pharmaceutical agent in situ,

-   -   thus causing the predetermined change to occur via said target        structure, wherein said predetermined change targets the        condition, disorder or disease.

In a different preferred embodiment, the present invention provides amethod for treating a condition, disorder or disease mediated by atarget structure in a subject, comprising:

(1) administering to the subject an activatable pharmaceutical agentthat is capable of activation by multi photon absorption and effecting apredetermined change in said target structure when activated bydifferent wavelengths of UV or visible or infrared irradiation emittedfrom at least one energy modulation agent in a vicinity of or within thetarget structure; and

(2) applying an initiation energy from an initiation energy source tothe subject,

wherein the initiation energy applied and activatable pharmaceuticalagent upon activation produce insufficient singlet oxygen in the subjectto produce cell lysis, and wherein the initiation energy activates theactivatable pharmaceutical agent by the multi photon absorption event insitu,

-   -   thus causing the predetermined change to occur via said target        structure, wherein said predetermined change targets the        condition, disorder or disease.

In a different preferred embodiment, the present invention provides amethod for treating a condition, disorder or disease mediated by atarget structure in a subject, comprising:

(1) administering to the subject an activatable pharmaceutical agentthat is capable of activation by multi photon absorption and effecting apredetermined change in said target structure when activated bydifferent wavelengths of UV or visible or infrared irradiation emittedfrom at least one energy modulation agent in a vicinity of or within thetarget structure; and

(2) applying an initiation energy from an initiation energy source tothe subject,

wherein the initiation energy applied and activatable pharmaceuticalagent upon activation produce a controlled amount of singlet oxygen inthe subject to produce cell lysis, and wherein the initiation energyactivates the activatable pharmaceutical agent by the multi photonabsorption event in situ,

-   -   thus causing the predetermined change to occur via said target        structure, wherein said predetermined change targets the        condition, disorder or disease.

Work in the area of photodynamic therapy has shown that the amount ofsinglet oxygen required to cause cell lysis, and thus cell death, is0.32×10⁻³ mol/liter or more, or 10⁹ singlet oxygen molecules/cell ormore. In one embodiment, it is preferable to avoid production of anamount of singlet oxygen that would cause cell lysis, due to itsindiscriminate nature of attack, lysing both target cells and healthycells. Accordingly, in one embodiment, the level of singlet oxygenproduction caused by the initiation energy used or activatablepharmaceutical agent upon activation is less than level needed to causecell lysis.

One advantage is that multiple wavelengths of emitted radiation may beused to selectively stimulate one or more photoactivatable agents orenergy modulation agents capable of stimulating the one or morephotoactivatable agents. The energy modulation agent is preferablystimulated at a wavelength and energy that causes little or no damage tohealthy cells, with the energy from one or more energy modulation agentsbeing transferred, such as by Foerster Resonance Energy Transfer, to thephotoactivatable agents that damage the cell and cause the onset of thedesired cellular change, e.g., apoptosis of the cells.

Another advantage is that side effects can be greatly reduced bylimiting the production of free radicals, singlet oxygen, hydroxides andother highly reactive groups that are known to damage healthy cells.Furthermore, additional additives, such as antioxidants, may be used tofurther reduce undesired effects of irradiation.

In a different embodiment, it is preferable to control the amount ofsinglet oxygen that would cause cell lysis relative to the amount ofactivated psoralen produced.

Accordingly, in this embodiment, the level of singlet oxygen productioncaused by the initiation energy used or activatable pharmaceutical agentupon activation is less than or equal to the amount of activatedpsoralen. For example, the amount of singlet oxygen produced can rangefrom 1% to 95% of the activated psorlen produced. In another example,the amount of singlet oxygen produced can range from 10% to 90% of theactivated psoralen produced. In another example, the amount of singletoxygen produced can range from 20% to 80% of the activated psorlenproduced. In another example, the amount of singlet oxygen produced canrange from 30% to 70% of the activated psoralen produced. In anotherexample, the amount of singlet oxygen produced can range from 40% to 60%of the activated psorlen produced.

In a different embodiment, it is preferable to control the amount ofsinglet oxygen to be more than or equal to the amount of activatedpsoralen. For example, the amount of activated psorlen produced canrange from 1% to 95% of the singlet oxygen produced. In another example,the amount of activated psoralen produced can range from 10% to 90% ofthe singlet oxygen produced. In another example, the amount of activatedpsorlen produced can range from 20% to 80% of the singlet oxygenproduced. In another example, the amount of activated psoralen producedcan range from 30% to 70% of the singlet oxygen produced. In anotherexample, the amount of activated psoralen produced can range from 40% to60% of the singlet oxygen produced.

Resonance Energy Transfer (RET) is an energy transfer mechanism betweentwo molecules having overlapping emission and absorption bands.Electromagnetic emitters are capable of converting an arrivingwavelength to a longer wavelength. For example, UV-B energy absorbed bya first molecule may be transferred by a dipole-dipole interaction to aUV-A-emitting molecule in close proximity to the UV-B-absorbingmolecule. Alternatively, a material absorbing a shorter wavelength maybe chosen to provide RET to a non-emitting molecule that has anoverlapping absorption band with the transferring molecule's emissionband. Alternatively, phosphorescence, chemiluminescence, orbioluminescence may be used to transfer energy to a photoactivatablemolecule.

Yet another example is that nanoparticles or nanoclusters of certainatoms may be introduced such that are capable of resonance energytransfer over comparatively large distances, such as greater than onenanometer, more preferably greater than five nanometers, even morepreferably at least 10 nanometers. Functionally, resonance energytransfer may have a large enough “Foerster” distance (R₀), such thatnanoparticles in one part of a cell are capable of stimulatingactivation of photoactivatable agents disposed in a distant portion ofthe cell, so long as the distance does not greatly exceed R₀. Forexample, gold nanospheres having a size of 5 atoms of gold have beenshown to have an emission band in the ultraviolet range, recently.

In one embodiment, an aggressive cell proliferation disorder has a muchhigher rate of mitosis, which leads to selective destruction of adisproportionate share of the malignant cells during even a systemicallyadministered treatment. Stem cells and healthy cells may be spared fromwholesale programmed cell death, even if exposed to photoactivatedagents, provided that such photoactivated agents degenerate from theexcited state to a lower energy state prior to binding, mitosis or othermechanisms for creating damage to the cells of a substantial fraction ofthe healthy stem cells. Thus, an auto-immune response may not beinduced.

Alternatively, a blocking agent may be used that prevents or reducesdamage to stem cells or healthy cells, selectively, which wouldotherwise be impaired. The blocking agent is selected or is administeredsuch that the blocking agent does not impart a similar benefit tomalignant cells, for example.

In one embodiment, stem cells are targeted, specifically, fordestruction with the intention of replacing the stem cells with a donorcell line or previously stored, healthy cells of the patient. In thiscase, no blocking agent is used. Instead, a carrier or photosensitizeris used that specifically targets the stem cells.

Any of the photoactivatable agents may be exposed to an excitationenergy source implanted in a subject preferably near a target site. Thephotoactive agent may be directed to a receptor site by a carrier havinga strong affinity for the receptor site. Within the context of thepresent invention, a “strong affinity” is preferably an affinity havingan equilibrium dissociation constant, K_(i), at least in the nanomolar,nM, range or higher. Preferably, the carrier may be a polypeptide andmay form a covalent bond with a photoactive agent, for example. Thepolypeptide may be an insulin, interleukin, thymopoietin or transferrin,for example. Alternatively, a photoactive agent may have a strongaffinity for the target cell without binding to a carrier.

A receptor site may be any of the following: nucleic acids of nucleatedblood cells, molecule receptor sites of nucleated blood cells, theantigenic sites on nucleated blood cells, epitopes, or other sites wherephotoactive agents are capable of destroying a targeted cell.

In one embodiment, thin fiber optic lines are inserted in the subjectand external light is used to photoactivate the agents. In anotherembodiment, a plurality of sources for supplying electromagneticradiation energy or energy transfer can be used.

The phenomenon of ultra weak emission from cellular systems has been atopic of various inquiries since the 1900s. This topic can be tracedback to the early investigations of the Russian biologist GurwitschAlexander G. Gurwitsch more than seventy years ago, who speculated thatultraweak photon emission transmit information in cells [A. G.Gurwitsch, S. S. Grabje, and S. Salkind, “Die Natur des spezifischenErregers der Zellteilung,” Arch. Entwicklungsmech. Org. 100, 11-40,1923].

In the 1970s, this area of research was investigated by a number ofinvestigators. The presence of biological radiation from a variety ofcells was later investigated by several research groups in Europe andJapan using low-noise, sensitive photon-counting detection systems [B.Ruth and F.-A. Popp, “Experimentelle Untersuchungen zur ultraschwachenPhotonenemission biologischer Systeme,” Z. Naturforsch., A: Phys. Sci.31c, 741-745, 1976; T. I. Quickenden and S. S. Que-Hee, “The spectraldistribution of the luminescence emitted during growth of the yeastSaccharomyces cerevisiae and its relationship to mitogenetic radiation,”Photochem. Photobiol. 23, 201-204, 1976; H. Inaba, Y. Shimizu, Y. Tsuji,and A. Yamagishi, “Photon counting spectral analysing system ofextra-weak chemi- and bioluminescence for biochemical applications,”Photochem. Photobiol. 30, 169-175, 1979]. Popp and coworkers suggestedthe evidence of some ‘informational character’ associated with theultra-weak photon emission from biological systems, often referred byPopp as “bio-photons”. Other studies reported ultra-weak photon emissionfrom various species including plant, and animals cells [H. J. Niggli,C. Scaletta, Y. Yan, F.-A. Popp, and L. A. Applegate, “Ultraweak photonemission in assessing bone growth factor efficiency using fibroblasticdifferentiation,” J. Photochem. Photobiol., B, 64, 62-68, 2001;].Results of experiments of UV-irradiated skin fibroblasts indicated thatrepair deficient xeroderma pigmentosum cells show an efficient increaseof ultraweak photon emission in contrast to normal cells. [H. J. Niggli,“Artificial sunlight irradiation induces ultraweak photon emission inhuman skin fibroblasts,” J. Photochem. Photobiol., B 18, 281-285(1993)].

A delayed luminescence emission was also observed in biological systems[F.-A. Popp and Y. Yan, “Delayed luminescence of biological systems interms of coherent states,” Phys. Lett. A 293, 93-97 (2002); A. Scordino,A. Triglia, F. Musumeci, F. Grasso, and Z. Rajfur, “Influence of thepresence of Atrazine in water on in-vivo delayed luminescence ofacetabularium acetabulum,” J. Photochem. Photobiol., B, 32, 11-17(1996); This delayed luminescence was used in quality control ofvegetable products [A. Triglia, G. La Malfa, F. Musumeci, C. Leonardi,and A. Scordino, “Delayed luminescence as an indicator of tomato fruitquality,” J. Food. Sci. 63, 512-515 (1998)] or for assessing the qualityor quality changes of biological tissues [Yu Yan, Fritz-Albert Popp*,Sibylle Sigrist, Daniel Schlesinger, Andreas Dolf, Zhongchen Yan, SophieCohen, Amodsen Chotia, “Further analysis of delayed luminescence ofplants”, Journal of Photochemistry and Photobiology B: Biology 78,235-244 (2005)].

It has been reported that UV excitation can further enhance theultra-weak emission and a method for detecting UV-A-laser-inducedultra-weak photon emission was used to evaluate differences betweencancer and normal cells. [H. J. Niggli et al,Laser-ultraviolet-A-induced ultraweak photon emission in mammaliancells, Journal of Biomedical Optics 10(2), 024006 (2005)].

Accordingly, in one embodiment of the present invention, upon applyingan initiation energy from at least one source to a target structure in asubject in need of treatment, the initiation energy contacts the targetstructure and induces a predetermined change in said target structure insitu by exposure of the target structure to different wavelengths of UVor visible or infrared irradiation emitted from at least one energymodulation agent in a vicinity of or within the target structure,

wherein the predetermined change is the enhancement of energy emissionfrom the target, which then mediates, initiates or enhances a biologicalactivity of other target structures in the subject, or of a second typeof target structure (e.g., a different cell type). Here, a firstwavelength would induce the predetermined change and a second wavelengthwould mediates, initiates or enhances a biological activity of othertarget structures in the subject, or of a second type of targetstructure (e.g., a different cell type).

In a further embodiment, a biocompatible emitting source, such as afluorescing metal nanoparticle or fluorescing dye molecule, is selectedthat emits in the UV-A band. The UV-A emitting source is directed to thesite of a disease or condition. The UV-A emitting source may be directedto the site of the disease or condition by systemically administeringthe UV-A emitting source. Preferably, the UV-A emitting source isconcentrated in the target site, such as by physical insertion or byconjugating the UV-A emitting molecule with a specific carrier that iscapable of concentrating the UV-A emitting source in a specific targetstructure, as is known in the art.

In one embodiment, the UV-A emitting source is a gold nanoparticlecomprising a cluster of 5 gold atoms, such as a water soluble quantumdot encapsulated by polyamidoamine dendrimers. The gold atom clustersmay be produced (for example according to procedures known in the art)through a slow reduction of gold salts (e.g. HAuCl₄ or AuBr₃) or otherencapsulating amines, for example. One advantage of such a goldnanoparticle is the increased Foerster distance (i.e. R₀), which may begreater than 100 angstroms.

In one embodiment of this invention, in addition to the UV-A emittingsource, a UV-B or UV-C emitting source is directed to the site of adisease or condition to act as a germicide. In one embodiment of thisinvention, in addition to the UV-A emitting source, a NIR emittingsource is directed to the site of a disease or condition to act as ananti-inflammatory or to promote cellular proliferation or to reducepain. A number of commercially available drugs described below couldalso be activated by the a NIR emitting source.

Porfimer sodium (Photofrin; QLT Therapeutics, Vancouver, BC, Canada), isa partially purified preparation of hematoporphyrin derivative (HpD).Photofrin has been approved by the US Food and Drug Administration forthe treatment of obstructing esophageal cancer, microinvasiveendobronchial non-small cell lung cancer, and obstructing endobronchialnon-small cell lung cancer. Photofrin is activated with 630 nm, whichhas a tissue penetration of approximately 2 to 5 mm. Photofrin has arelatively long duration of skin photosensitivity (approximately 4 to 6weeks).

Tetra (m-hydroxyphenyl) chlorin (Foscan; Scotia Pharmaceuticals,Stirling, UK), is a synthetic chlorine compound that is activated by 652nm light. Clinical studies have demonstrated a tissue effect of up to 10mm with Foscan and 652 nm light. Foscan is more selectively aphotosensitizer in tumors than normal tissues, and requires acomparatively short light activation time. A recommended dose of 0.1mg/kg is comparatively low and comparatively low doses of light may beused. Nevertheless, duration of skin photosensitivity is reasonable(approximately 2 weeks). However, Foscan induces a comparatively highyield of singlet oxygen, which may be the primary mechanism of DNAdamage for this molecule.

Motexafin lutetium (Lutetium texaphryin) is activated by light in thenear infared region (732 nm). Absorption at this wavelength has theadvantage of potentially deeper penetration into tissues, compared withthe amount of light used to activate other photosensitizers (FIGS. 2Aand 2B). Lutetium texaphryin also has one of the greatest reportedselectivities for tumors compared to selectivities of normal tissues.Young S W, et al.: Lutetium texaphyrin (PCI-0123) a near-infrared,water-soluble photosensitizer. Photochem Photobiol 1996, 63:892-897. Inaddition, its clinical use is associated with a shorter duration of skinphotosensitivity (24 to 48 hours). Lutetium texaphryin has beenevaluated for metastatic skin cancers. It is currently underinvestigation for treatment of recurrent breast cancer and for locallyrecurrent prostate cancer. The high selectivity for tumors promisesimproved results in clinical trials.

While the description of the invention describes specific examples usingnanoparticles, the present invention in many embodiments is not limitedto particles having a size less than one micron. However, in many of theembodiments, the size range of having a size less than one micron, andespecially less than 100 nm produces properties of special interest suchas for example emission lifetime luminescence quenching, luminescentquantum efficiency, and concentration quenching and such as for examplediffusion, penetration, and dispersion into mediums where larger sizeparticles would not migrate.

In one embodiment, similar to that described above, the first wavelengthwould induce the predetermined change and the second wavelength wouldmediates, initiates or enhances neuronal spike firing

For example, a light-sensitive protein (for example, channelrhodopsin-2(ChR2) and chloride pump halorhodopsin (NpHR)) can be incorporated intothe lentiviral vector or other vector providing delivery of thelight-sensitive protein encoding gene into a target cell. ChR2containing a light sensor and a cation channel, provides electricalstimulation of appropriate speed and magnitude to activate neuronalspike firing, when the cells harboring Ch2R are pulsed with light.

In one embodiment for use as either the first or second wavelength, alanthanide chelate capable of intense luminescence is used. For example,a lanthanide chelator may be covalently joined to a coumarin or coumarinderivative or a quinolone or quinolone-derivative sensitizer.Sensitizers may be a 2- or 4-quinolone, a 2- or 4-coumarin, orderivatives or combinations of these examples. A carbostyril 124(7-amino-4-methyl-2-quinolone), a coumarin 120(7-amino-4-methyl-2-coumarin), a coumarin 124(7-amino-4-(trifluoromethyl)-2-coumarin), aminoinethyltrimethylpsoralenor other similar sensitizer may be used. Chelates may be selected toform high affinity complexes with lanthanides, such as terbium oreuropium, through chelator groups, such as DTPA. Such chelates may becoupled to any of a wide variety of well known probes or carriers, andmay be used for resonance energy transfer to a psoralen orpsoralen-derivative, such as 8-MOP, or other photoactive moleculescapable of binding DNA. In one alternative example, the lanthanidechelate is localized at the site of the disease using an appropriatecarrier molecule, particle or polymer, and a source of electromagneticenergy is introduced by minimally invasive procedures to irradiate thetarget structure, after exposure to the lanthanide chelate and aphotoactive molecule.

In another embodiment for use as either the first or second wavelength,a biocompatible, endogenous fluorophore emitter is selected to stimulateresonance energy transfer to a photoactivatable molecule. Abiocompatible emitter with an emission maxima within the absorptionrange of the biocompatible, endogenous fluorophore emitter may beselected to stimulate an excited state in fluorophore emitter. One ormore halogen atoms may be added to any cyclic ring structure capable ofintercalation between the stacked nucleotide bases in a nucleic acid(either DNA or RNA) to confer new photoactive properties to theintercalator. Any intercalating molecule (psoralens, coumarins, or otherpolycyclic ring structures) may be selectively modified by halogenationor addition of non-hydrogen bonding ionic substituents to impartadvantages in its reaction photochemistry and its competitive bindingaffinity for nucleic acids over cell membranes or charged proteins, asis known in the art.

In general, any source for activation of the pharmaceutical agent, suchas electrical, chemical and/or radiation, can be used individually orcombined into a system for activating an activatable molecule. Theprocess may be a photopheresis process or may be similar tophotophoresis. While photophoresis is generally thought to be limited tophotonic excitation, such as by UV-light, other forms of radiation maybe used as a part of a system to activate an activatable molecule.Radiation includes ionizing radiation which is high energy radiation,such as an X-ray or a gamma ray, which interacts to produce ion pairs inmatter. Radiation also includes high linear energy transfer irradiation,low linear energy transfer irradiation, alpha rays, beta rays, neutronbeams, accelerated electron beams, and ultraviolet rays. Radiation alsoincludes proton, photon and fission-spectrum neutrons. Higher energyionizing radiation may be combined with chemical processes to produceenergy states favorable for resonance energy transfer, for example.Other combinations and variations of these sources of excitation energymay be combined as is known in the art, in order to stimulate theactivation of an activatable molecule, such as 8-MOP. In one example,ionizing radiation is directed at a solid tumor and stimulates, directlyor indirectly, activation of 8-MOP, as well as directly damaging the DNAof malignant tumor cells. In this example, either the effect of ionizingradiation or the photophoresis-like activation of 8-MOP may be thoughtof as an adjuvant therapy to the other.

In one embodiment, the present invention provides a method for treatinga condition, disease or disorder mediated by a target structure in asubject, comprising:

-   -   (1) administering to the subject an activatable pharmaceutical        agent that is capable of effecting a predetermined change when        activated by exposure of the target structure to different        wavelengths of UV or visible or infrared irradiation emitted        from at least one energy modulation agent in a vicinity of or        within the target structure; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,        wherein the initiation energy source is a source of energy        capable of penetrating completely through the subject, and        wherein the applying activates the activatable agent in situ,    -   thus causing the predetermined change to occur, wherein        occurrence of the predetermined change in the target structure        causes an increase in rate or decrease in rate of cell division        and/or growth to treat the condition, disease or disorder.

In a further embodiment, the present invention provides a method fortreating a condition, disease or disorder mediated by a target structurein a subject, comprising:

(1) administering to the subject one or more energy modulation agentsand an activatable pharmaceutical agent that is capable of effecting apredetermined change in the target structure when activated by exposureof the target structure to different wavelengths of UV or visible orinfrared irradiation emitted from at least one energy modulation agentin a vicinity of or within the target structure; and

(2) applying an initiation energy from an initiation energy source tothe subject,

wherein the one or more energy modulation agents convert the initiationenergy applied to UV-A or visible energy, which then activates theactivatable agent in situ,

-   -   thus causing the predetermined change to occur, wherein        occurrence of the predetermined change causes an increase in        rate or decrease in rate of cell division and/or growth to treat        the condition, disease or disorder.

In a different embodiment, the activatable pharmaceutical agent can beactivated by a single or multiphoton absorption event.

In yet another embodiment, the activatable pharmaceutical agent,preferably a photoactive agent, is directed to a receptor site by acarrier having a strong affinity for the receptor site. The carrier maybe a polypeptide and may form a covalent bond with a photo active agent,for example. The polypeptide may be an insulin, interleukin,thymopoietin or transferrin, for example. Alternatively, a photoactivepharmaceutical agent may have a strong affinity for the target cellwithout a binding to a carrier.

For example, a treatment may be applied that acts to slow or pausemitosis. Such a treatment is capable of slowing the division of rapidlydividing healthy cells or stem cells without pausing mitosis ofcancerous cells. Thus, the difference in growth rate between thenon-target cells and target cells are further differentiated to enhancethe effectiveness of the methods of the present invention.

In a further embodiment, methods in accordance with the presentinvention may further include adding an additive to alleviate treatmentside-effects. Exemplary additives may include, but are not limited to,antioxidants, adjuvant, or combinations thereof. In one exemplaryembodiment, psoralen is used as the activatable pharmaceutical agent,UV-A is used as the activating energy, and antioxidants are added toreduce the unwanted side-effects of irradiation.

In another aspect, the present invention also provides methods forproducing an autovaccine, including: (1) providing a population oftargeted cells; (2) treating the cells ex vivo with a psoralen or aderivative thereof; (3) activating the psoralen with an initiationenergy source to induce a predetermined change in a target structure inthe population of the target cells by exposure of the target structureto different wavelengths of UV or visible or infrared irradiationemitted from at least one energy modulation agent in a vicinity of orwithin the target structure; and (4) returning the treated cells back tothe host to induce an autovaccine effect against the targeted cell,wherein the treated cells cause an autovaccine effect.

In a different embodiment, a method for generating an autovaccine for asubject, comprises:

-   -   (1) providing a population of target cells;    -   (2) treating the target cells ex vivo in an environment separate        and isolated from the subject with an activatable pharmaceutical        agent capable of activation by a multi photon absorption event;    -   (3) exposing the treated target cells to an energy source;    -   (4) activating the activatable pharmaceutical agent with the        energy source by the multi photon absorption event to induce a        predetermined change in at least one target structure in the        target cells by exposure of the target cells to different        wavelengths of UV or visible or infrared irradiation emitted        from at least one energy modulation agent in a vicinity of or        within the target cell; and    -   (5) returning the thus changed cells back to the subject to        induce in the subject an autovaccine effect against the target        cells.

In a further embodiment, methods in accordance with the presentinvention may further include a method for modifying a target structurewhich mediates or is associated with a biological activity, comprising:

-   -   (1) contacting said target structure with at least one        activatable pharmaceutical agent that is capable of effecting a        predetermined change in a target structure when activated and at        least one plasmonics-active agent; and    -   (2) applying an initiation energy from an initiation energy        source to target structure to expose the target structure to        different wavelengths of UV or visible or infrared irradiation        emitted from at least one energy modulation agent in a vicinity        of or within the target structure,    -   wherein the plasmonics-active agent enhances or modulates the        applied initiation energy, such that the enhanced initiation        energy activates the activatable agent    -   thus causing the predetermined change to the target structure to        occur, wherein said predetermined change modifies the target        structure and modulates the biological activity of the target        structure.

In a different embodiment, the predetermined change enhances theexpression of, promotes the growth of, or increases the quantity of saidtarget structure; enhances, inhibits or stabilizes the usual biologicalactivity of said target structure compared to a similar untreated targetstructure, and/or alters the immunological or chemical properties ofsaid target structure. In a different embodiment, said target structureis a compound that is modified by said predetermined change to be moreor less antigenic or immunogenic

The activatable pharmaceutical agent and derivatives thereof as well asthe energy modulation agent, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the activatable pharmaceutical agent and a pharmaceuticallyacceptable carrier. The pharmaceutical composition also comprises atleast one additive having a complementary therapeutic or diagnosticeffect, wherein the additive is one selected from an antioxidant, anadjuvant, or a combination thereof.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions. Modifications can be made to the compound of thepresent invention to affect solubility or clearance of the compound.These molecules may also be synthesized with D-amino acids to increaseresistance to enzymatic degradation. If necessary, the activatablepharmaceutical agent can be co-administered with a solubilizing agent,such as cyclodextran.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, rectal administration, and direct injection into theaffected area, such as direct injection into a tumor. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerin, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates, and agents for the adjustment of tonicity suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

In one embodiment of the invention, there is provided a pharmaceuticalcomposition for modifying a target structure. The pharmaceuticalcomposition includes at least one agent selected from the groupconsisting of energy modulation agents, plasmonics-active agents andcombinations thereof. The energy modulation agents includes one or morelight emitters capable of emitting at least two different wavelengths oflight, each wavelength of light associated with a different biologicalresponse, and the different wavelengths capable of activating differentbiological responses. The pharmaceutical composition preferably includesa pharmaceutically acceptable carrier.

Methods of administering agents according to the present invention arenot limited to the conventional means such as injection or oralinfusion, but include more advanced and complex forms of energytransfer. For example, genetically engineered cells that carry andexpress energy modulation agents may be used. Cells from the host may betransfected with genetically engineered vectors that expressbioluminescent agents. Transfection may be accomplished via in situ genetherapy techniques such as injection of viral vectors or gene guns, ormay be performed ex vivo by removing a sample of the host's cells andthen returning to the host upon successful transfection.

Such transfected cells may be inserted or otherwise targeted at the sitewhere diseased cells are located. In this embodiment, the initiationenergy source may be a biochemical source as such ATP, in which case theinitiation energy source is considered to be directly implanted in thetransfected cell. Alternatively, a conventional micro-emitter devicecapable of acting as an initiation energy source may be transplanted atthe site of the diseased cells.

It will also be understood that the order of administering the differentagents is not particularly limited. Thus in some embodiments theactivatable pharmaceutical agent may be administered before the energymodulation agent, while in other embodiments the energy modulation agentmay be administered prior to the activatable pharmaceutical agent. Itwill be appreciated that different combinations of ordering may beadvantageously employed depending on factors such as the absorption rateof the agents, the localization and molecular trafficking properties ofthe agents, and other pharmacokinetics or pharmacodynamicsconsiderations.

A further embodiment is the use of the present invention for thetreatment of skin cancer. In this example, a photoactivatable agent,preferably psoralen, is given to the patient, and is delivered to theskin lesion via the blood supply. An activation source having limitedpenetration ability (such as UV or IR) is shined directly on the skin—inthe case of psoralen, it would be a UV light, or an IR source. With theuse of an IR source, the irradiation would penetrate deeper and generateUV via two single photon events with psoralen. The skin lesion isexposed to different wavelengths of UV or visible or infraredirradiation emitted from at least one energy modulation agent in avicinity of or within the skin lesion.

In a further embodiment, methods according to this aspect of the presentinvention further include a step of separating the components of thetreated cells into fractions and testing each fraction for autovaccineeffect in a host. The components thus isolated and identified may thenserve as an effective autovaccine to stimulate the host's immune systemto suppress growth of the targeted cells.

In another aspect, the present invention further provides systems andkits for practicing the above described methods.

In one embodiment, a system for producing an auto-vaccine in a subject,comprises:

-   -   at least one activatable pharmaceutical agent that is capable of        activation by a multiphoton absorption event and of inducing a        predetermined cellular change via at least one target structure        in a target cell in said subject by exposure of the target        structure to different wavelengths of UV or visible or infrared        irradiation emitted from at least one energy modulation agent in        a vicinity of or within the target structure;    -   means for placing said at least one activatable pharmaceutical        agent in said subject; and    -   an initiation energy source to provide initiation energy capable        of activating the at least one activatable pharmaceutical agent        in said target cell by the multi photon absorption event,        wherein activation is either direct or indirect.

In a different embodiment, a system in accordance with the presentinvention may include: (1) an initiation energy source; and (2) one ormore energy modulation agents. The system may further comprise (3) oneor more activatable pharmaceutical agents. In an additional embodiment,the system may comprise only (1) the initiation energy source. In yetanother embodiment, the system may comprise (1) an initiation energysource; and (3) one or more activatable pharmaceutical agents. FIG. 3illustrates a system according to one exemplary embodiment of thepresent invention. Referring to FIG. 3 , an exemplary system accordingto one embodiment of the present invention may have an initiation energysource 1 directed at the subject 4. An activatable pharmaceutical agent2 and an energy modulation agent 3 are administered to the subject 4.The initiation energy source may additionally be controlled by acomputer system 5 that is capable of directing the delivery of theinitiation energy.

In preferred embodiments, the initiation energy source may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. One example of such linear accelerators is the SmartBeam™IMRT (intensity modulated radiation therapy) system from Varian medicalsystems (Varian Medical Systems, Inc., Palo Alto, Calif.).

In other embodiments, endoscopic or laproscopic devices equipped withappropriate initiation energy emitter may be used as the initiationenergy source. In such systems, the initiation energy may be navigatedand positioned at the pre-selected coordinate to deliver the desiredamount of initiation energy to the site.

In further embodiments, dose calculation and robotic manipulationdevices may also be included in the system.

In yet another embodiment, there is also provided a computer implementedsystem for designing and selecting suitable combinations of initiationenergy source, energy transfer agent, and activatable pharmaceuticalagent, comprising:

a central processing unit (CPU) having a storage medium on which isprovided:

-   -   a database of excitable compounds;    -   a first computation module for identifying and designing an        excitable compound that is capable of binding with a target        cellular structure or component; and    -   a second computation module predicting the different wavelengths        of UV or visible or infrared irradiation to be emitted from at        least one energy modulation agent in a vicinity of or within the        target cellular structure,

wherein the system, upon selection of a target cellular structure orcomponent, computes an excitable compound that is capable of bindingwith the target structure followed by a computation to predict theresonance absorption energy of the excitable compound.

FIG. 4 illustrates an exemplary computer implemented system according tothis embodiment of the present invention. Referring to FIG. 4 , anexemplary computer-implemented system according to one embodiment of thepresent invention may have a central processing unit (CPU) connected toa memory unit, configured such that the CPU is capable of processinguser inputs and selecting a combination of initiation source,activatable pharmaceutical agent, and energy transfer agent based on anenergy spectrum comparison for use in a method of the present invention.

FIG. 5 illustrates a computer system 1201 for implementing variousembodiments of the present invention. The computer system 1201 may beused as the controller 55 to perform any or all of the functions of theCPU described above. The computer system 1201 includes a bus 1202 orother communication mechanism for communicating information, and aprocessor 1203 coupled with the bus 1202 for processing the information.The computer system 1201 also includes a main memory 1204, such as arandom access memory (RAM) or other dynamic storage device (e.g.,dynamic RAM (DRAM), static RAM (SRAM), and synchronous DRAM (SDRAM)),coupled to the bus 1202 for storing information and instructions to beexecuted by processor 1203. In addition, the main memory 1204 may beused for storing temporary variables or other intermediate informationduring the execution of instructions by the processor 1203. The computersystem 1201 further includes a read only memory (ROM) 1205 or otherstatic storage device (e.g., programmable ROM (PROM), erasable PROM(EPROM), and electrically erasable PROM (EEPROM)) coupled to the bus1202 for storing static information and instructions for the processor1203.

The computer system 1201 also includes a disk controller 1206 coupled tothe bus 1202 to control one or more storage devices for storinginformation and instructions, such as a magnetic hard disk 1207, and aremovable media drive 1208 (e.g., floppy disk drive, read-only compactdisc drive, read/write compact disc drive, compact disc jukebox, tapedrive, and removable magneto-optical drive). The storage devices may beadded to the computer system 1201 using an appropriate device interface(e.g., small computer system interface (SCSI), integrated deviceelectronics (IDE), enhanced-IDE (E-IDE), direct memory access (DMA), orultra-DMA).

The computer system 1201 may also include special purpose logic devices(e.g., application specific integrated circuits (ASICs)) or configurablelogic devices (e.g., simple programmable logic devices (SPLDs), complexprogrammable logic devices (CPLDs), and field programmable gate arrays(FPGAs)).

The computer system 1201 may also include a display controller 1209coupled to the bus 1202 to control a display 1210, such as a cathode raytube (CRT), for displaying information to a computer user. The computersystem includes input devices, such as a keyboard 1211 and a pointingdevice 1212, for interacting with a computer user and providinginformation to the processor 1203. The pointing device 1212, forexample, may be a mouse, a trackball, or a pointing stick forcommunicating direction information and command selections to theprocessor 1203 and for controlling cursor movement on the display 1210.In addition, a printer may provide printed listings of data storedand/or generated by the computer system 1201.

The computer system 1201 performs a portion or all of the processingsteps of the invention (such as for example those described in relationto FIG. 5 ) in response to the processor 1203 executing one or moresequences of one or more instructions contained in a memory, such as themain memory 1204. Such instructions may be read into the main memory1204 from another computer readable medium, such as a hard disk 1207 ora removable media drive 1208. One or more processors in amulti-processing arrangement may also be employed to execute thesequences of instructions contained in main memory 1204. In alternativeembodiments, hard-wired circuitry may be used in place of or incombination with software instructions. Thus, embodiments are notlimited to any specific combination of hardware circuitry and software.

As stated above, the computer system 1201 includes at least one computerreadable medium or memory for holding instructions programmed accordingto the teachings of the invention and for containing data structures,tables, records, or other data described herein. Examples of computerreadable media are compact discs, hard disks, floppy disks, tape,magneto-optical disks, PROMs (EPROM, EEPROM, flash EPROM), DRAM, SRAM,SDRAM, or any other magnetic medium, compact discs (e.g., CD-ROM), orany other optical medium, punch cards, paper tape, or other physicalmedium with patterns of holes, a carrier wave (described below), or anyother medium from which a computer can read.

Stored on any one or on a combination of computer readable media, thepresent invention includes software for controlling the computer system1201, for driving a device or devices for implementing the invention,and for enabling the computer system 1201 to interact with a human user(e.g., print production personnel). Such software may include, but isnot limited to, device drivers, operating systems, development tools,and applications software. Such computer readable media further includesthe computer program product of the present invention for performing allor a portion (if processing is distributed) of the processing performedin implementing the invention.

The computer code devices of the present invention may be anyinterpretable or executable code mechanism, including but not limited toscripts, interpretable programs, dynamic link libraries (DLLs), Javaclasses, and complete executable programs. Moreover, parts of theprocessing of the present invention may be distributed for betterperformance, reliability, and/or cost.

The term “computer readable medium” as used herein refers to any mediumthat participates in providing instructions to the processor 1203 forexecution. A computer readable medium may take many forms, including butnot limited to, non-volatile media, volatile media, and transmissionmedia. Non-volatile media includes, for example, optical, magneticdisks, and magneto-optical disks, such as the hard disk 1207 or theremovable media drive 1208. Volatile media includes dynamic memory, suchas the main memory 1204. Transmission media includes coaxial cables,copper wire and fiber optics, including the wires that make up the bus1202. Transmission media also may also take the form of acoustic orlight waves, such as those generated during radio wave and infrared datacommunications.

Various forms of computer readable media may be involved in carrying outone or more sequences of one or more instructions to processor 1203 forexecution. For example, the instructions may initially be carried on amagnetic disk of a remote computer. The remote computer can load theinstructions for implementing all or a portion of the present inventionremotely into a dynamic memory and send the instructions over atelephone line using a modem. A modem local to the computer system 1201may receive the data on the telephone line and use an infraredtransmitter to convert the data to an infrared signal. An infrareddetector coupled to the bus 1202 can receive the data carried in theinfrared signal and place the data on the bus 1202. The bus 1202 carriesthe data to the main memory 1204, from which the processor 1203retrieves and executes the instructions. The instructions received bythe main memory 1204 may optionally be stored on storage device 1207 or1208 either before or after execution by processor 1203.

The computer system 1201 also includes a communication interface 1213coupled to the bus 1202. The communication interface 1213 provides atwo-way data communication coupling to a network link 1214 that isconnected to, for example, a local area network (LAN) 1215, or toanother communications network 1216 such as the Internet. For example,the communication interface 1213 may be a network interface card toattach to any packet switched LAN. As another example, the communicationinterface 1213 may be an asymmetrical digital subscriber line (ADSL)card, an integrated services digital network (ISDN) card or a modem toprovide a data communication connection to a corresponding type ofcommunications line. Wireless links may also be implemented. In any suchimplementation, the communication interface 1213 sends and receiveselectrical, electromagnetic or optical signals that carry digital datastreams representing various types of information.

The network link 1214 typically provides data communication through oneor more networks to other data devices. For example, the network link1214 may provide a connection to another computer through a localnetwork 1215 (e.g., a LAN) or through equipment operated by a serviceprovider, which provides communication services through a communicationsnetwork 1216. The local network 1214 and the communications network 1216use, for example, electrical, electromagnetic, or optical signals thatcarry digital data streams, and the associated physical layer (e.g., CAT5 cable, coaxial cable, optical fiber, etc). The signals through thevarious networks and the signals on the network link 1214 and throughthe communication interface 1213, which carry the digital data to andfrom the computer system 1201 maybe implemented in baseband signals, orcarrier wave based signals. The baseband signals convey the digital dataas unmodulated electrical pulses that are descriptive of a stream ofdigital data bits, where the term “bits” is to be construed broadly tomean symbol, where each symbol conveys at least one or more informationbits. The digital data may also be used to modulate a carrier wave, suchas with amplitude, phase and/or frequency shift keyed signals that arepropagated over a conductive media, or transmitted as electromagneticwaves through a propagation medium. Thus, the digital data may be sentas unmodulated baseband data through a “wired” communication channeland/or sent within a predetermined frequency band, different thanbaseband, by modulating a carrier wave. The computer system 1201 cantransmit and receive data, including program code, through thenetwork(s) 1215 and 1216, the network link 1214, and the communicationinterface 1213. Moreover, the network link 1214 may provide a connectionthrough a LAN 1215 to a mobile device 1217 such as a personal digitalassistant (PDA) laptop computer, or cellular telephone.

The exemplary energy spectrum previously noted in FIG. 1 may also beused in this computer-implemented system.

The reagents and chemicals useful for methods and systems of the presentinvention may be packaged in kits to facilitate application of thepresent invention. In one exemplary embodiment, a kit including apsoralen, and fractionating containers for easy fractionation andisolation of autovaccines is contemplated. A further embodiment of kitwould comprise at least one activatable pharmaceutical agent capable ofcausing a predetermined cellular change upon exposure of a targetstructure to different wavelengths of UV or visible or infraredirradiation emitted from at least one energy modulation agent in avicinity of or within the target structure, at least one energymodulation agent capable of activating the at least one activatableagent when energized, and containers suitable for storing the agents instable form, and preferably further comprising instructions foradministering the at least one activatable pharmaceutical agent and atleast one energy modulation agent to a subject, and for applying aninitiation energy from an initiation energy source to activate theactivatable pharmaceutical agent. The instructions could be in anydesired form, including but not limited to, printed on a kit insert,printed on one or more containers, as well as electronically storedinstructions provided on an electronic storage medium, such as acomputer readable storage medium. Also optionally included is a softwarepackage on a computer readable storage medium that permits the user tointegrate the information and calculate a control dose, to calculate andcontrol intensity of the irradiation source.

In different aspect of the invention, a kit for modifying a targetstructure which mediates or is associated with a biological activity,comprising:

-   -   at least one agent selected from the group consisting of energy        modulation agents, plasmonics-active agents and combinations        thereof;        -   wherein the energy modulation agent, if present, upgrades or            downgrades an initiation energy to an activation energy            capable of causing, either directly or indirectly, a            predetermined change in the target structure upon exposure            of the target structure to different wavelengths of UV or            visible or infrared irradiation emitted from at least one            energy modulation agent in a vicinity of or within the            target structure;        -   wherein the plasmonics-active agent, if present, enhances or            modifies the applied initiation energy or the activation            energy generated by the energy modulation agent, or both;            and    -   one or more containers suitable for storing the agents in stable        forms.

In a different embodiment, a kit for performing a condition, disorder ordisease treatment, comprises:

at least one energy modulation agent capable of adsorbing, intensifyingor modifying an initiation energy into an energy that is capable ofcausing a predetermined change in a target structure upon exposure of atarget structure to different wavelengths of UV or visible or infraredirradiation emitted from at least one energy modulation agent in avicinity of or within the target structure; and

containers suitable for storing the agents in stable form.

In yet another embodiment, the kit may further comprise instructions foradministering the at least one energy modulation agent to a subject.

In various embodiments of the present invention, the plasmonics-enhancedspectroscopic properties (spectral absorption, emission, scattering) canbe involved in the treatment.

The plasmonics-enhanced spectroscopic (PEPST) principle is based on theenhancement mechanisms of the electromagnetic field effect. There aretwo main sources of electromagnetic enhancement: (1) first, the laserelectromagnetic field is enhanced due to the addition of a field causedby the polarization of the metal particle; (2) in addition to theenhancement of the excitation laser field, there is also anotherenhancement due to the molecule radiating an amplified emission(luminescence, Raman, etc.) field, which further polarizes the metalparticle, thereby acting as an antenna to further amplify theRaman/Luminescence signal.

Electromagnetic enhancements are divided into two main classes: a)enhancements that occur only in the presence of a radiation field, andb) enhancements that occur even without a radiation field. The firstclass of enhancements is further divided into several processes. Plasmaresonances on the substrate surfaces, also called surface plasmons,provide a major contribution to electromagnetic enhancement. Aneffective type of plasmonics-active substrate comprises nanostructuredmetal particles, protrusions, or rough surfaces of metallic materials.Incident light irradiating these surfaces excites conduction electronsin the metal, and induces excitation of surface plasmons leading toRaman/luminescence enhancement. At the plasmon frequency, the metalnanoparticles (or nanostructured roughness) become polarized, resultingin large field-induced polarizations and thus large local fields on thesurface. These local fields increase the luminescence/Raman emissionintensity, which is proportional to the square of the applied field atthe molecule. As a result, the effective electromagnetic fieldexperienced by the analyte molecule on these surfaces is much largerthan the actual applied field. This field decreases as 1/r³ away fromthe surface. Therefore, in the electromagnetic models, theluminescence/Raman-active analyte molecule is not required to be incontact with the metallic surface but can be located anywhere within therange of the enhanced local field, which can polarize this molecule. Thedipole oscillating at the wavelength λ of Raman or luminescence can, inturn, polarize the metallic nanostructures and, if λ is in resonancewith the localized surface plasmons, the nanostructures can enhance theobserved emission light (Raman or luminescence).

There are two main sources of electromagnetic enhancement: (1) first,the laser electromagnetic field is enhanced due to the addition of afield caused by the polarization of the metal particle; (2) in additionto the enhancement of the excitation laser field, there is also anotherenhancement due to the molecule radiating an amplifiedRaman/luminescence field, which further polarizes the metal particle,thereby acting as an antenna to further amplify the Raman/luminescencesignal. Plasmonics-active metal nanoparticles also exhibit stronglyenhanced visible and near-infrared light absorption, several orders ofmagnitude more intense compared to conventional laser phototherapyagents. The use of plasmonic nanoparticles as highly enhancedphotoabsorbing agents thus provides a selective and efficientphototherapy strategy. The tunability of the spectral properties of themetal nanoparticles and the biotargeting abilities of the plasmonicnanostructures make the PEPST method promising.

PEPST is based on several important mechanisms:

-   -   Increased absorption of the excitation light by the plasmonic        metal nanoparticles, resulting in enhanced photoactivation of        drug molecules    -   Increased absorption of the excitation light by the plasmonic        metal nanoparticles that serve as more efficient energy        modulation agent systems, yielding more light for increased        excitation of PA molecules    -   Increased absorption of the excitation light by the photoactive        drug system adsorbed on or near the plasmonic metal        nanoparticles    -   Increased light absorption of the energy modulation agent        molecules adsorbed on or near the metal nanoparticles    -   Amplified light emission from the energy modulation agent        molecules adsorbed on or near the metal nanoparticles    -   Increased absorption of emission light emitted from the energy        modulation agent by the PA molecule

Experimental evidence suggests that the origin of the 10⁶- to 10¹⁵-foldRaman enhancement primarily arises from two mechanisms: a) anelectromagnetic “lightning rod” effect occurring near metal surfacestructures associated with large local fields caused by electromagneticresonances, often referred to as “surface plasmons”; and b) a chemicaleffect associated with direct energy transfer between the molecule andthe metal surface.

According to classical electromagnetic theory, electromagnetic fieldscan be locally amplified when light is incident on metal nanostructures.These field enhancements can be quite large (typically 10⁶- to 10⁷-fold,but up to 10¹⁵-fold enhancement at “hot spots”). When a nanostructuredmetallic surface is irradiated by an electromagnetic field (e.g., alaser beam), electrons within the conduction band begin to oscillate ata frequency equal to that of the incident light. These oscillatingelectrons, called “surface plasmons,” produce a secondary electric fieldwhich adds to the incident field. If these oscillating electrons arespatially confined, as is the case for isolated metallic nanospheres orroughened metallic surfaces (nanostructures), there is a characteristicfrequency (the plasmon frequency) at which there is a resonant responseof the collective oscillations to the incident field. This conditionyields intense localized field enhancements that can interact withmolecules on or near the metal surface. In an effect analogous to a“lightning rod,” secondary fields are typically most concentrated atpoints of high curvature on the roughened metal surface.

Design, Fabrication and Operation of PEPST Probes

FIGS. 7A-7G show a number of the various embodiments of PEPST probesthat can be designed:

-   -   (A) probe comprising PA molecules bound to a metal (gold)        nanoparticle;    -   (B) PA-containing nanoparticle covered with metal nanoparticles;    -   (C) Metal nanoparticle covered with PA nanocap;    -   (D) PA-containing nanoparticle covered with metal nanocap;    -   (E) Metal nanoparticle covered with PA nanoshell;    -   (F) PA-containing nanoparticle covered with metal nanoshell; and    -   (G) PA-containing nanoparticle covered with metal nanoshell with        protective coating layer.

A basic embodiment of the PEPST probe is shown in FIG. 7A. This probecomprises PA molecules bound to a metal (e.g., gold) nanoparticle. FIGS.8A-8B illustrate the plasmonics-enhancement effect of the PEPST probe.The gold nanoparticles can serve as a drug delivery platform. Goldnanoparticles have been described as a novel technology in the field ofparticle-based tumor-targeted drug delivery [Giulio F. Paciotti andLonnie Myer, DavidWeinreich, Dan Goia, Nicolae Pavel, Richard E.McLaughlin, Lawrence Tamarkin, “Colloidal Gold: A Novel NanoparticleVector for Tumor Directed Drug Delivery, Drug Delivery, 11:169-183,2004]. Particle delivery systems capable of escaping phagocyticclearance by the reticuloendothelial system (RES) can facilitatetargeting cancer therapeutics to solid tumors. Such delivery systemscould preferentially accumulate within the tumor microenvironment underideal conditions. A particle delivery system capable of sequestering aphototherapeutic drug selectively within a tumor may also reduce theaccumulation of the drug in healthy organs. Consequently, these deliverysystems may increase the relative efficacy or safety of therapy (lessradiation energy and intensity), and therefore, will increase the drug'stherapeutic efficiency.

Radiation of suitable energy is used to excite the PA drug molecules(e.g., aminolevulinic acid (ALA), porphyrins) and make them photoactive.For example, with the PDT drug ALA, light of a HeNe laser (632.8-nmexcitation) can be used for excitation. In this case the metalnanoparticles are designed to exhibit strong plasmon resonance bandaround 632.8 nm. The surface plasmon resonance effect amplifies theexcitation light at the nanoparticles, resulting in increasedphotoactivation of the PA drug molecules and improved therapyefficiency. The plasmonics-enhanced mechanism can also be used with theother PEPST probes in FIGS. 7B, 7C, 7D, 7E, 7F and 7G. FIGS. 34A-34Gshow yet another embodiment of plasmonics photo-active probes. FIGS.35A-35G show yet another embodiment of plasmonics photo-active probesthat have a dielectric layer between the metal and the UC materials.

In one embodiment, a method for treating a condition, disorder ordisease in accordance with the present invention comprises:

-   -   (1) administering to the subject at least one activatable        pharmaceutical agent that is capable of effecting a        predetermined change in a target structure when activated and at        least one plasmonics-active agent; and    -   (2) applying an initiation energy from an initiation energy        source to the subject,    -   wherein the plasmonics-active agent enhances or modifies the        applied initiation energy, such that the enhanced initiation        energy activates the activatable agent in situ,    -   thus causing the predetermined change to the target structure to        occur, wherein said predetermined change modifies the target        structure and treats said condition, disorder, or disease.

In a different embodiment, a method in accordance with the presentinvention comprises:

-   -   (1) contacting said target structure with at least one        activatable pharmaceutical agent that is capable of effecting a        predetermined change in a target structure when activated and at        least one plasmonics-active agent; and    -   (2) applying an initiation energy from an initiation energy        source to target structure to expose the target structure to        different wavelengths of UV or visible or infrared irradiation        emitted from at least one energy modulation agent in a vicinity        of or within the target structure    -   wherein the plasmonics-active agent enhances or modifies the        applied initiation energy, such that the enhanced initiation        energy activates the activatable agent    -   thus causing the predetermined change to the target structure to        occur, wherein said predetermined change modifies the target        structure and modulates the biological activity of the target        structure.

In a different embodiment, at least one energy modulation agent and/orexcitation-generating energy modulation agent material may be alsoadded. In one embodiment, the energy modulation agent orexcitation-generating energy modulation agent material may adsorb,intensify or modify the initiation energy which is then enhanced by atleast one plasmonic agent. In a different embodiment, the energymodulation agent or excitation-generating energy modulation agentmaterial may adsorb, intensify or modify energy enhanced by the at leastplasmonics-active agent and emit an energy that is capable to activatethe pharmaceutical activatable agent.

In another embodiment, the predetermined change enhances the expressionof, promotes the growth of, or increases the quantity of said targetstructure. In yet, different embodiment, the predetermined changeenhances, inhibits or stabilizes the usual biological activity of saidtarget structure compared to a similar untreated target structure. In adifferent embodiment, the predetermined change alters the immunologicalor chemical properties of said target structure. In a differentembodiment, the target structure is a compound that is modified by saidpredetermined change to be more or less antigenic or immunogenic.

Structures of Plasmonics-Active Metal Nanostructures

These shells typically comprise a metallic layer over a dielectric core.In one embodiment of the present invention, shells comprising a metalliclayer over a dielectric core can be spheroidal shells. The plasmonresonances (both longitudinal and transverse modes) are influenced byboth shell thickness and aspect ratio. The present invention alsoincludes prolate and oblate spheroidal shells, which show someinteresting qualitative features in their plasmon resonances. Thespheroidal shell presents two degrees of freedom for tuning: the shellthickness and the shell aspect ratio.

FIGS. 9A-9J show some of the various embodiments of plasmonics-activenanostructures that can be designed, and are preferred embodiments ofthe present invention:

-   -   (A) Metal nanoparticle;    -   (B) Dielectric nanoparticle core covered with metal nanocap;    -   (C) Spherical metal nanoshell covering dielectric spheroid core;    -   (D) Oblate metal nanoshell covering dielectric spheroid core;    -   (E) Metal nanoparticle core covered with dielectric nanoshell;    -   (F) Metal nanoshell with protective coating layer;    -   (G) Multi layer metal nanoshells covering dielectric spheroid        core;    -   (H) Multi-nanoparticle structures;    -   (I) Metal nanocube and nanotriangle/nanoprism; and    -   (J) Metal cylinder.

In a further embodiment of the present invention, the PA drug moleculescan be incorporated into a material (e.g., biocompatible polymer) thatcan form a nanocap onto the metal (gold) nanoparticles. The material canbe a gel or biocompatible polymer that can have long-term continuousdrug release properties. Suitable gel or biocompatible polymers include,but are not limited to poly(esters) based on polylactide (PLA),polyglycolide (PGA), polycarpolactone (PCL), and their copolymers, aswell as poly(hydroxyalkanoate)s of the PHB-PHV class, additionalpoly(ester)s, natural polymers, particularly, modifiedpoly(saccharide)s, e.g., starch, cellulose, and chitosan, polyethyleneoxides, poly(ether)(ester) block copolymers, and ethylene vinyl acetatecopolymers. The drug release mechanism can also be triggered bynon-invasive techniques, such as RF, MW, ultrasound, photon (FIG. 10 ).

FIGS. 11A-11C show other possible embodiments where the PA drug moleculeis bound to the metal nanoparticles via a linker that can be cut by aphoton radiation. Such a linker includes, but is not limited to, abiochemical bond (FIG. 11A), a DNA bond (FIG. 11B), or anantibody-antigen bond (FIG. 11C). In another embodiment, the linker is achemically labile bond that will be broken by the chemical environmentinside the cell. These types of probes are useful for therapy modalitieswhere the PA molecules have to enter the nucleus (e.g., psoralenmolecules need to enter the nucleus of cells and intercalate onto DNA).Since it is more difficult for metal nanoparticles to enter the cellnucleus than for smaller molecules, it is desirable to PEPST probes thathave releasable PA molecules.

Aggregation of metal (such as silver or gold) nanoparticles(nanospheres, nanorods, etc) is often a problem, especially withcitrate-capped gold nanospheres, cetyl trimethylammonium bromide(CTAB)-capped gold nanospheres and nanorods and nanoshells because theyhave poor stability when they are dispersed in buffer solution due tothe aggregating effect of salt ions. The biocompatibility can beimproved and nanoparticle aggregation prevented by capping thenanoparticles with polyethylene glycol (PEG) (by conjugation ofthiol-functionalized PEG with metal nanoparticles). Furthermore,PEGylated nanoparticles are preferentially accumulated into tumortissues due to the enhanced permeability and retention effect, known asthe “EPR.” Blood vessels in tumor tissue are more “leaky” than in normaltissue, and as a result, particles, or large macromolecular species orpolymeric species preferentially extravasate into tumor tissue.Particles and large molecules tend to stay a longer time in tumor tissuedue to the decreased lymphatic system, whereas they are rapidly clearedout in normal tissue. This tumor targeting strategy is often referred toas passive targeting whereas the antibody-targeting strategy is calledactive targeting.

To specifically target diseased cells, specific genes or proteinmarkers, the drug systems of the present invention can be bound to abioreceptor (e.g., antibody, synthetic molecular imprint systems, DNA,proteins, lipids, cell-surface receptors, aptamers, etc.).Immunotargeting modalities to deliver PA agents selectively to thediseased cells and tissue provide efficient strategies to achievingspecificity, minimizing nonspecific injury to healthy cells, andreducing the radiation intensity used. Biofunctionalization of metalnanoparticles (e.g., gold, silver) can be performed using commonlydeveloped and widely used procedures. There are several targetingstrategies that can be used in the present invention: (a) nanoparticlesconjugated to antibodies that recognize biomarkers specific to thediseased cells; (b) nanoparticles passivated by poly (ethylene) glycol(PEG), which is used to increase the biocompatibility and biostabilityof nanoparticles and impart them an increased blood retention time.

Bioreceptors are the key to specificity for targeting disease cells,mutated genes or specific biomarkers. They are responsible for bindingthe biotarget of interest to the drug system for therapy. Thesebioreceptors can take many forms and the different bioreceptors thathave been used are as numerous as the different analytes that have beenmonitored using biosensors. However, bioreceptors can generally beclassified into five different major categories. These categoriesinclude: 1) antibody/antigen, 2) enzymes, 3) nucleic acids/DNA, 4)cellular structures/cells and 5) biomimetic. FIGS. 12A-12G illustrate anumber of embodiments of the various PEPSI probes with bioreceptors thatcan be designed.

Antibody Probes. Antibody based targeting is highly active, specific andefficient. The antibodies are selected to target a specific tumor marker(e.g., anti-epidermal growth factor receptor (EGFR) antibodies targetedagainst overexpressed EGFR on oral and cervical cancer cells; anti-Her2antibodies against overexpressed Her2 on breast cancer cells) Antibodiesare biological molecules that exhibit very specific binding capabilitiesfor specific structures. This is very important due to the complexnature of most biological systems. An antibody is a complex biomolecule,made up of hundreds of individual amino acids arranged in a highlyordered sequence. For an immune response to be produced against aparticular molecule, a certain molecular size and complexity arenecessary: proteins with molecular weights greater than 5000 Da aregenerally immunogenic. The way in which an antigen and itsantigen-specific antibody interact may be understood as analogous to alock and key fit, by which specific geometrical configurations of aunique key enables it to open a lock. In the same way, anantigen-specific antibody “fits” its unique antigen in a highly specificmanner. This unique property of antibodies is the key to theirusefulness in immunosensors where only the specific analyte of interest,the antigen, fits into the antibody binding site.

DNA Probes. The operation of gene probes is based on the hybridizationprocess. Hybridization involves the joining of a single strand ofnucleic acid with a complementary probe sequence. Hybridization of anucleic acid probe to DNA biotargets (e.g., gene sequences of amutation, etc) offers a very high degree of accuracy for identifying DNAsequences complementary to that of the probe. Nucleic acid strands tendto be paired to their complements in the corresponding double-strandedstructure. Therefore, a single-stranded DNA molecule will seek out itscomplement in a complex mixture of DNA containing large numbers of othernucleic acid molecules. Hence, nucleic acid probe (i.e., gene probe)detection methods are very specific to DNA sequences. Factors affectingthe hybridization or reassociation of two complementary DNA strandsinclude temperature, contact time, salt concentration, and the degree ofmismatch between the base pairs, and the length and concentration of thetarget and probe sequences.

Biologically active DNA probes can be directly or indirectly immobilizedonto a drug system, such as the energy modulation agent system (e.g.,gold nanoparticle, a semiconductor, quantum dot, a glass/quartznanoparticles, etc.) surface to ensure optimal contact and maximumbinding. When immobilized onto gold nanoparticles, the gene probes arestabilized and, therefore, can be reused repetitively. Several methodscan be used to bind DNA to different supports. The method commonly usedfor binding DNA to glass involves silanization of the glass surfacefollowed by activation with carbodiimide or glutaraldehyde. Thesilanization methods have been used for binding to glass surfaces using3 glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane(APTS), followed by covalently linking DNA via amino linkersincorporated either at the 3′ or 5′ end of the molecule during DNAsynthesis.

Enzyme Probes. Enzymes are often chosen as bioreceptors based on theirspecific binding capabilities as well as their catalytic activity. Inbiocatalytic recognition mechanisms, the detection is amplified by areaction catalyzed by macromolecules called biocatalysts. With theexception of a small group of catalytic ribonucleic acid molecules, allenzymes are proteins. Some enzymes require no chemical groups other thantheir amino acid residues for activity. Others require an additionalchemical component called a cofactor, which may be either one or moreinorganic ions, such as Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, or a more complexorganic or metalloorganic molecule called a coenzyme. The catalyticactivity provided by enzymes allows for much lower limits of detectionthan would be obtained with common binding techniques. The catalyticactivity of enzymes depends upon the integrity of their native proteinconformation. If an enzyme is denatured, dissociated into its subunits,or broken down into its component amino acids, its catalytic activity isdestroyed. Enzyme-coupled receptors can also be used to modify therecognition mechanisms.

In one embodiment, nanoparticles of metal colloid hydrosols are preparedby rapidly mixing a solution of AgNO₃ with ice-cold NaBH₄. Fordeveloping a SMP probes, a DNA segment is bound to a nanoparticle ofsilver or gold. The immobilization of biomolecules (e.g., DNA,antibodies, enzymes, etc.) to a solid support through covalent bondsusually takes advantage of reactive groups such as amine (—NH₂) orsulfide (—SH) that naturally are present or can be incorporated into thebiomolecule structure. Amines can react with carboxylic acid or estermoieties in high yield to form stable amide bonds. Thiols canparticipate in maleimide coupling yielding stable dialkylsulfides.

In one embodiment, silver nanoparticles are used. In one embodiment, theimmobilization schemes involving Ag surfaces utilize a priorderivatization of the surface with alkylthiols, forming stable linkagesare used. Alkylthiols readily form self-assembled monolayers (SAM) ontosilver surfaces in micromolar concentrations. The terminus of thealkylthiol chain can be directly used to bind biomolecules, or can beeasily modified to do so. The length of the alkylthiol chain was foundto be an important parameter, keeping the biomolecules away from thesurface. Furthermore, to avoid direct, non-specific DNA adsorption ontothe surface, alkylthiols were used to block further access to thesurface, allowing only covalent immobilization through the linker.

Silver/gold surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 15°. There is also a larger thiol packing density on silver,when compared to gold.

After SAM formation on silver nanoparticles, alkylthiols can becovalently coupled to biomolecules. The majority of synthetic techniquesfor the covalent immobilization of biomolecules utilize free aminegroups of a polypeptide (enzymes, antibodies, antigens, etc) or ofamino-labeled DNA strands, to react with a carboxylic acid moietyforming amide bonds. In one embodiment, more active intermediate (labileester) is first formed with the carboxylic acid moiety and in a laterstage reacted with the free amine, increasing the coupling yield.Successful coupling procedures include:

The coupling approach used to bind DNA to a silver nanoparticle involvesthe esterification under mild conditions of a carboxylic acid with alabile group, an N-hydroxysuccinimide (NHS) derivative, and furtherreaction with free amine groups in a polypeptide (enzymes, antibodies,antigens, etc) or amine-labeled DNA, producing a stable amide [4]. NHSreacts almost exclusively with primary amine groups. Covalentimmobilization can be achieved in as little as 30 minutes. Since H₂Ocompetes with —NH₂ in reactions involving these very labile esters, itis important to consider the hydrolysis kinetics of the available estersused in this type of coupling. The derivative of NHS used in FIG. 101 ,O—(N-succinimidyl)-N,N,N,N′-tetramethyluronium tetrafluoroborate,increases the coupling yield by utilizing a leaving group that isconverted to urea during the carboxylic acid activation, hence favorablyincreasing the negative enthalpy of the reaction.

Photon Excitation in the Therapeutic Window of Tissue

There are several methods using light to excite photoactivate compoundsnon-invasively with the use of light having wavelengths within theso-called “therapeutic window” (700-1300 nm). The ability of light topenetrate tissues depends on absorption. Within the spectral range knownas the therapeutic window (or diagnostic window), most tissues aresufficiently weak absorbers to permit significant penetration of light.This window extends from 600 to 1300 nm, from the orange/red region ofthe visible spectrum into the NIR. At the short-wavelength end, thewindow is bound by the absorption of hemoglobin, in both its oxygenatedand deoxygenated forms. The absorption of oxygenated hemoglobinincreases approximately two orders of magnitude as the wavelengthshortens in the region around 600 nm. At shorter wavelengths many moreabsorbing biomolecules become important, including DNA and the aminoacids tryptophan and tyrosine. At the infrared (IR) end of the window,penetration is limited by the absorption properties of water. Within thetherapeutic window, scattering is dominant over absorption, and so thepropagating light becomes diffuse, although not necessarily enteringinto the diffusion limit. FIG. 13 shows a diagram of the therapeuticwindow of tissue. The following section discusses the use of one-photonand multi-photon techniques for therapy.

Light Excitation Methods: Single-Photon and Multi-Photon Excitation

Two methods can be used, one-photon or multi-photon excitation. If thetwo-photon technique is used, one can excite the PA molecules with lightat 700-1000 nm, which can penetrate deep inside tissue, in order toexcite molecules that absorb in the 350-500 nm spectral region. Thisapproach can excite the psoralen compounds, which absorb in the 290-350nm spectral region and emit in the visible. With the one-photon method,the photo-activator (PA) drug molecules can directly absorb excitationlight at 600-1300 nm. In this case we can design a psoralen-relatedsystem (e.g., psoralens having additional aromatic rings or otherconjugation to alter the ability to absorb at different wavelengths) oruse other PA systems: photodynamic therapy drugs, ALA, etc.

PEPST Modality for Photopheresis Using X Ray Excitation

Need for X-Ray Excitation

Photopheresis has been demonstrated to be an effective treatment for anumber of diseases. One method for an improved and practical modalityfor such therapy was described in U.S. Ser. No. 11/935,655, filed Nov.6, 2007, the entire contents of which are hereby incorporated byreference.

Although X-ray can excite compounds in deep tissue non-invasively, X-rayis not easily absorbed by organic drug compounds. The present inventionprovides a solution to that problem, by the providing of a molecularsystem that can absorb the X-ray energy and change that energy intoother energies that can be used to activate drug molecules. Morespecifically, the molecular system that can absorb and change the X-rayenergy in the present invention is the PEPST probes comprisingnanoparticles.

In this embodiment, the present invention uses X-rays for excitation.The advantage is the ability to excite molecules non-invasively sinceX-ray can penetrate deep in tissue. However, the limitation is the factthat X-ray does not interact with most molecules. In one embodiment ofthe present invention, the drug molecule (or PA) is bound to a molecularentity, referred to as an “energy modulation agent” that can interactwith the X-rays, and then emit light that can be absorbed by the PA drugmolecules. (FIG. 14 )

PEPST Probes for X Ray Excitation

The dose delivered to a tumor during photon-based radiation therapy canbe enhanced by loading high atomic number (Z) materials such as gold(Au, Z=79) into the tumor, resulting in greater photoelectric absorptionwithin the tumor than in surrounding tissues. Thus, gold clearly leadsto a higher tumor dose than either iodine or gadolinium. Second,nanoparticles provide a better mechanism than microspheres, in terms ofdelivering high-Z materials to the tumor, overcoming some of thedifficulties found during an earlier attempt using gold microspheres

Gold (or metal) complexes with PA ligands: Gold (or metal) complexeswith PA can preferably be used in the present invention. The metal canbe used as an energy modulation agent system. For example, goldcomplexes with psoralen-related ligands can be used as a hybrid energymodulation agent-PA system. The gold molecules serve as the energymodulation agent system and the ligand molecules serve as the PA drugsystem. Gold(I) complexes with diphosphine and bipyridine ligandsexhibit X-ray excited luminescence

FIGS. 15A-15F show a number of the various embodiments of PEPST probesthat can be preferably used for X ray excitation of energy modulationagent-PA system. These probes comprise:

-   -   (A) PA molecules bound to energy modulation agent and to        plasmonic metal nanoparticle;    -   (B) Plasmonic metal nanoparticle with energy modulation agent        nanocap covered with PA molecules;    -   (C) PA-covered nanoparticle with plasmonic metal nanoparticles;    -   (D) Energy modulation agent-containing nanoparticle covered with        PA molecules and plasmonic metal nanocap;    -   (E) Plasmonic metal nanoparticle core with energy modulation        agent nanoshell covered with PA molecule; and    -   (F) PA molecule bound to energy modulation agent (attached to        plasmonics metal nanoparticle) nanoparticle by detachable        biochemical bond.        Examples of PEPST System Based on Energy Modulation Agent-PA

For purposes of simplification, the following discussion is centered ongold as the metal material and CdS as the energy modulation agentmaterial (which can also be used as DNA stabilized CdS, see Ma et al,Langmuir, 23 (26), 12783-12787 (2007)) and psoralen as the PA molecule.However, it is to be understood that many other embodiments of metalmaterial, energy modulation agent and PA molecule are possible withinthe bounds of the present invention, and the following discussion is forexemplary purposes only. Suitable metals that can be used in plasmonresonating shells or other plasmon resonating structures can be include,but are not limited to, gold, silver, platinum, palladium, nickel,ruthenium, rhenium, copper, and cobalt.

In the embodiment of FIG. 15A, the PEPST system comprises goldnanoparticles, an energy modulation agent nanoparticle (e.g., CdS)linked to a PA drug molecule (e.g., psoralen). X ray is irradiated toCdS, which absorbs X rays and emits CdS XEOL light (at 350-400 nm) thatis plasmonics-enhanced by the gold nanoparticle. This enhanced XEOLlight is used to photoactivate psoralen (PA molecule). In this case thenanostructure of the gold nanoparticle is designed to enhance the XEOLlight at 350-400 nm.

In the embodiment of FIG. 15B, the PEPST system comprises aplasmonics-active metal (gold) nanoparticle with energy modulation agentnanocap (CdS) covered with PA molecules (e.g., psoralen). X ray isirradiated to CdS, which absorbs X ray and emits XEOL light that isplasmonics-enhanced by the gold nanoparticle. This enhanced XEOL lightis used to photoactivate psoralen (PA molecule).

In the embodiment of FIG. 15C, the PEPST system comprises a PA (e.g.,psoralen)-covered CdS nanoparticle with smaller plasmonic metal (gold)nanoparticles. X ray is irradiated to CdS, which absorbs X ray and emitsXEOL light that is plasmonics-enhanced by the gold nanoparticle. Thisenhanced XEOL light is used to photoactivate psoralen (PA molecule).

In the embodiment of FIG. 15D, the energy modulation agent corecomprises CdS or CsCl nanoparticles covered with a nanocap of gold. Xray is irradiated to CdS or CsCl, which absorbs X ray and emits XEOLlight that is plasmonics-enhanced by the gold nanocap structure. Thisenhanced XEOL light is used to photoactivate psoralen (PA molecule).

Similarly, the embodiment in FIG. 15E comprises a spherical gold corecovered by a shell of CdS or CsCl. X ray is irradiated to CdS or CsClmaterial, which absorbs X ray and emits XEOL light that isplasmonics-enhanced by the gold nanosphere. This enhanced XEOL light isused to photoactivate psoralen (PA molecule).

In the embodiment of FIG. 15F, the PEPST system comprises goldnanoparticles, and an energy modulation agent nanoparticle (e.g., CdS)linked to a PA drug molecule (e.g., psoralen) by a link that can bedetached by radiation. X ray is irradiated to CdS, which absorbs X rayand emits CdS XEOL light (at 350-400 nm) that is plasmonics-enhanced bythe gold nanoparticle. This enhanced XEOL light is used to photoactivatepsoralen (PA molecule). In this case the nanostructure of the goldnanoparticle is designed to enhance the XEOL light at 350-400 nm.

In alternative embodiments, the metal nanoparticles or single nanoshellsare replaced by multi layers of nanoshells

In other alternative embodiments the metal nanoparticles are coveredwith a layer (1-30 nm) of dielectric material (e.g. silica). Thedielectric layer (or nanoshell) is designed to prevent quenching of theluminescence light emitted by the energy modulation agent (also referredto as EEC) molecule(s) due to direct contact of the metal with theenergy modulation agent molecules. In yet other alternative embodiments,the energy modulation agent molecules or materials are bound to (or inproximity of) a metal nanoparticle via a spacer (linker). The spacer isdesigned to prevent quenching of the luminescence light emitted by theenergy modulation agent molecules or materials.

Other Useable Materials

The energy modulation agent materials can include any materials that canabsorb X ray and emit light in order to excite the PA molecule. Theenergy modulation agent materials include, but are not limited to:

metals (gold, silver, etc);

quantum dots;

semiconductor materials;

scintillation and phosphor materials;

materials that exhibit X-ray excited luminescence (XEOL);

organic solids, metal complexes, inorganic solids, crystals, rare earthmaterials (lanthanides), polymers, scintillators, phosphor materials,etc.; and

materials that exhibit excitonic properties.

Quantum dots, semiconductor nanostructures. Various materials related toquantum dots, semiconductor materials, etc. can be used as energymodulation agent systems. For example CdS-related nanostructures havebeen shown to exhibit X-ray excited luminescence in the UV and/orvisible region. For example, ZnO nanoparticles or quantum-dots could beused for excited luminescence in the UV and/or visible region.

Scintillator Materials as energy modulation agent systems. Variousscintillator materials can be used as energy modulation agents sincethey absorb X-ray and emit luminescence emission, which can be used toexcite the PA system. For example, single crystals of molybdates can beexcited by X-ray and emit luminescence around 400 nm

Solid Materials as energy modulation agent systems: Various solidmaterials can be used as energy modulation agents due to their X-rayexcited luminescence properties. For example CdS (or CsCl) exhibitluminescence when excited by soft X-ray.

XEOL materials can include lanthanides or rare earth materials Someexamples of metal complexes exhibiting XEOL which can be used as energymodulation agent systems are shown in FIGS. 16A-16B and 17 . Suchstructures can be modified by replacing the metal atom with metalnanoparticles in order to fabricate a plasmonics-enhance PEPST probe. Inthe present invention, the experimental parameters including size, shapeand metal type of the nano structure can be selected based upon theexcitation radiation (NIR or X ray excitation), the photoactivationradiation (UVB), and/or the emission process from the energy modulationagent system (visible NIR).

U.S. Pat. No. 7,008,559 (the entire contents of which are incorporatedherein by reference) describes the upconversion performance of ZnS whereexcitation at 767 nm produces emission in the visible range. Thematerials described in U.S. Pat. No. 7,008,559 (including the ZnS aswell as Er³⁺ doped BaTiO₃ nanoparticles and Yb³⁺ doped CsMnCl₃) aresuitable in various embodiments of the invention.

Further materials suitable as energy modulation agents include, but arenot limited to, CdTe, CdSe, ZnO, CdS, Y₂O₃, MgS, CaS, SrS and BaS. Suchmaterials may be any semiconductor and more specifically, but not by wayof limitation, sulfide, telluride, selenide, and oxide semiconductorsand their nanoparticles, such as Zn_(1-x)Mn_(x)S_(y),Zn_(1-x)Mn_(x)Se_(y), Zn_(1-x)Mn_(x)Te_(y), Cd_(1-x)MnS_(y),Cd_(1-x)Mn_(x)Se_(y), Cd_(1-x)Mn_(x)Te_(y), Pb_(1-x)Mn_(x)S_(y),Pb_(1-x)Mn_(x)Se_(y), Pb_(1-x)Mn_(x)Te_(y), Mg_(1-x)MnSy,Ca_(1-x)Mn_(x)S_(y), Ba_(1-x)Mn_(x)S_(y) and Sr_(1-x), etc. (wherein,0<x≤1, and 0<y≤1). Complex compounds of the above-describedsemiconductors are also contemplated for use in the invention—e.g.(M_(1-z)N_(z))_(1-x)Mn_(x)A_(1-y)By (M=Zn, Cd, Pb, Ca, Ba, Sr, Mg; N═Zn,Cd, Pb, Ca, Ba, Sr, Mg; A=S, Se, Te, O; B═S, Se, Te, O; 0<x≤1, 0<y≤1,0<z≤1). Two examples of such complex compounds areZn_(0.4)Cd_(0.4)Mn_(0.2)S and Zn_(0.9)Mn_(0.1)S_(0.8)Se_(0.2).Additional energy modulation materials include insulating andnonconducting materials such as BaF₂, BaFBr, and BaTiO₃, to name but afew exemplary compounds. Transition and rare earth ion co-dopedsemiconductors suitable for the invention include sulfide, telluride,selenide and oxide semiconductors and their nanoparticles, such as ZnS;Mn; Er; ZnSe; Mn, Er; MgS; Mn, Er; CaS; Mn, Er; ZnS; Mn, Yb; ZnSe; Mn,Yb; MgS; Mn, Yb; CaS; Mn, Yb etc., and their complex compounds:(M_(1-z)N_(z))_(1-x)(Mn_(q)R_(1-q))_(x)A_(1-y)B_(y) (M=Zn, Cd, Pb, Ca,Ba, Sr, Mg; N═Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S, Se, Te, O; B═S, . . .0<z<1, o<q<1). Further materials suitable as energy modulation agentsinclude LaPO₄: Ce, Tb and 3Ca₃ (PO₄)₂ Ca(Fl, Cl)₂:Sb³⁺, Mn²⁺.

Some nanoparticles such as ZnS:Tb³⁺, Er³⁺; ZnS:Tb³⁺; Y₂O₃:Tb³⁺;Y₂O₃:Tb³⁺, Er3⁺; ZnS:Mn²⁺; ZnS:Mn,Er³⁺ are known in the art to functionfor both down-conversion luminescence and upconversion luminescence, andcan thus be used in various embodiments of the present invention.

These and the other energy modulation agents described herein can becoated with a passivation or biocompatible material to protect it fromits environment (e.g., the biological medium of a human or animal body)and vice versa to protect its environment from the energy modulationagent. In some embodiments, the biocompatible material is abiocompatible, non-toxic layer as shown in FIG. 34 . In someembodiments, the biocompatible material covering the energy modulationagents is a biocompatible polymer that can form a nanocap. Thebiocompatible material can be a gel or biocompatible polymer. Suitablegel or biocompatible polymers include, but are not limited topoly(esters) based on polylactide (PLA), polyglycolide (PGA),polycarpolactone (PCL), and their copolymers, as well aspoly(hydroxyalkanoate)s of the PHB-PHV class, additional poly(ester)s,natural polymers, particularly, modified poly(saccharide)s, e.g.,starch, cellulose, and chitosan, polyethylene oxides, poly(ether)(ester)block copolymers, and ethylene vinyl acetate copolymers.

In some embodiments, the biocompatible material can be nano-diamond filmor diamond like carbon coating or a highly conductive graphene material.A wide variety of diamond like carbon (DCL) coating materials are knownin the art, ranging in sp³ to sp² concentrations and including a varietyof dopants or impurities, especially hydrogen. The range of sp³ to sp²concentrations and the concentration of dopants or impurities influencesthe optical, electrical, and mechanical properties of the resulting DLCcoating. In the literature, the phase diagram in FIG. 37 isrepresentative.

As demonstration, a variety of these DLC materials for theircompatibility as a biocompatible coating and suitable UV transmittance.There appears to be a wide diversity of thicknesses and ranges of Hconcentration and sp³ to sp² concentrations, which are suitable here asa coating.

In one example, a mixture of two phosphors (LaPO₄: Ce, Tb and 3Ca₃(PO₄)₂ Ca(Fl, Cl)₂:Sb³⁺, Mn²⁺) were coated with ethyl cellulose (EC),coated with a predominantly sp² DLC (SP2), and were coated with anhydrogenated DLC film (H100). FIG. 36 is a plot of relative cell killusing the various coatings as compared to a control sample having nophosphor. The results show that all of these films had a substantiallyhigh fraction of cell kill when exposed to x-ray irradiation at thex-ray peak voltage sand currents listed. On average, all the coatedsamples seemed to have a higher fractional cell kill than the uncoatedphosphors, indicating that the coatings did not substantially obscurethe emitted light from the phosphors.

Diamond and Diamond-Like Carbon Energy Modulation Agents

The use of a diamond material (diamond crystals, nano-diamond films, ormicron to sub-micron size diamond particles or DLC) as an energymodulation agent is also possible in the present invention.

U.S. Pat. No. 7,927,390 (the entire contents of which are incorporatedherein by reference) describes aqueous suspensions of finely divideddiamond particles which would be suitable sources of diamond for thepresent invention. As noted therein, these suspensions could be forexample an aqueous suspension liquid of finely divided diamond particlescomprising 0.05 to 160 parts by weight of a finely divided diamondparticles in 1000 parts of water, with the finely divided diamondparticles having an element composition consisting mainly of 72 to 89.5%by weight of carbon, 0.8 to 1.5% of hydrogen, 1.5 to 2.5% of nitrogen,and 10.5 to 25.0% of oxygen. These diamond particles would havediameters in the range of 2 nm to 50 nm in diameters thereof (80% ormore by number average, 70% or more by weight average). although largerand smaller diamond particles could be used.

U.S. Pat. No. 5,087,434 (the entire contents of which are incorporatedherein by reference) describes compositions of synthetic diamondparticles and their synthesis by homogeneous nucleation of seedparticles in the gas phase followed by growth of diamond or diamond-likecarbon on these seeds. The techniques and diamond particles used in thispatent are applicable here for the energy modulation agents of thisinvention.

Presently fluorescent nanodiamond particles are commercially availablefrom Adamas Nanotechnologies, Inc. of Raleigh, N.C. These materials aresuitable for the diamond based energy modulation agents of thisinvention. The fluoresence of nanodiamond particles is based on colorcenters incorporated into the diamond lattice. Nitrogen-vacancy centers(N-V) provide red fluorescence and N-V-N (or H3 centers) emit greenlight. These nanodiamond particles are biocompatibile, permit largediamond surface area (per volume), and permit surface functionalization.

NV centers in ND can be in a number of ways. In on approach, NDsmanufactured by static high-pressure, high-temperature (HPHT) synthesisand containing about 100 ppm of substitutional N, are irradiated with40-keV He+ ions followed by annealing. See Chang, Y.-R. et al. NatureNanotechnology, 2008, 3, 284, the entire contents of which areincorporated herein by reference.

Besides diamond, diamond like carbon is available. U.S. Pat. No.6,265,068 (the entire contents of which are incorporated herein byreference) describes inorganic phosphor particles having a diamond-likecarbon coating and the method of making these particles. In this patent,the inorganic phosphor particles were coated with a diamond-like carboncoating in a plasma system. The coatings and phosphors in that patentare suitable for coating the energy modulation agents of this invention.The coated phosphors of that patent would be suitable as energymodulation agents of this invention.

In various embodiments of this invention, diamond or diamond like carbonexcited by deep UV light, x-rays, e-beams, and other energetic particlesemits light in the ultraviolet and/or visible range which in turnexcites the PA molecules or materials attached (e.g., tethered to) orremoved from the diamond.

In early reports of visible luminescence, diamond was made to luminescein a variety of ways including by irradiating with ultra-violet rays,cathode rays or X-rays or by the action of heat or friction. Workersreported that diamonds, which fluoresced blue in ultra-violet rays, alsofluoresced blue in X-rays; but the color in X-rays had less of a violetcolor. Workers also reported that diamonds not emitting diamonds underUV exposure, nevertheless luminesced under X-rays although weakly. Inthis invention, the use of ultra-violet rays, cathode rays or X-rayswould be more preferred, than that of heat or friction.

Defects in the diamond and impurities in the diamond are known to affectthe emitted wavelength. Known defects and/or dopants include nitrogen,boron, hydrogen, oxygen, silicon, phosphorus, nickel, cobalt, sulfur,manganese, tungsten, and iron. Of these, boron dopants can induce bluephosphorescence under UV light exposure. Yellow and green emissions arepossible with nickel impurities. A number of “intrinsic” defects (i.e.,the displacement of carbon atoms from their normal crystallographicposition) have also been recognized.

With deep UV excitation at 223 nm, broad emissions are seen at about 428and 470 nm, corresponding to photon energies 2.90 and 2.64 eV,respectively. As shown in FIG. 34H, the broad emissions extend into theultraviolet.

Similar emissions are observed for electron-induced orcathodoluminescence. The observed emissions are dependent on the dopantsor impurities in the diamond. Under 20 kV electrons, a number ofcathodoluminescent peaks have been observed. A dominant emission isobserved at 430 nm and with several weak emissions occurring at 480,500, 560, and 740 nm. The dominant emission is associated with a nearestneighbor donor acceptor recombination. The emission at 500 and 600 nm isassociated with the nitrogen. The emission at 740 nm is associated withsilicon. A representative set of spectra of chemical vapor depositeddiamond is shown in FIG. 341 . FIG. 34J shows the luminescent spectraobtained from higher energy electron irradiation at 100 kV. A number ofdeep UV emissions in the 200 to 250 nm range were observed. Thenanodiamonds discussed above from Adamas Nanotechnologies have prominentvisible emissions depending on the dopant or impurity, and would beexpected to have deep UV emission lines also. See FIG. 34K.

Besides the diamond discussed above, there have been reports ofdiamond-like carbon compounds also showing photoluminescence under deepUV 325 nm excitations. In particular, molybdenum-containing diamond-likecarbon (Mo-DLC) thin films have been synthesized which have-MoCnanocrystallites with sizes of 1-2 nm embedded in amorphous carboncross-linked structures. These Mo-DLC films showed a photoluminescence(PL) band in blue with the PL peak divided into two bands with the peakpositions at about 405 and 455 nm.

Thus, in various embodiments of the invention, diamond or diamond-likecarbon materials can be used as both a coating on the other energymodulation agents or separately as the primary energy modulation agent.For example, the quantum dots noted above can be coated or decoratedwith diamond or diamond-like carbon material.

In a further embodiment, a diamond or diamond-like carbon material canbe used as the energy modulation agent, such that the diamond ordiamond-like carbon material has a plurality of defect types present inthe same material, such that upon excitation, a single material emitsmultiple wavelengths of light. Alternatively, a plurality of diamond ordiamond-like carbon materials can be used as energy modulation agents,such that the plurality of materials each have different defects presentthan the other of the plurality of materials, enabling the fine tuningof light emissions in a plurality of wavelengths as desired. Thisprovides the capability to configure unique energy modulation agentswhere excitation (by electrons, x-rays, or deep UV) of the compositediamond or diamond-like carbon material on or in the presence of othersof the energy modulation agents noted above such that a plurality ofdistinct wavelengths can be emitted from the composite structure. In oneembodiment, emissions from either of the diamond or diamond-like carbonmaterial and/or the non-diamond energy modulation agent can producesecondary emissions from each other. In one embodiment, emissions fromeither of the diamond or diamond-like carbon material and/or thenon-diamond energy modulation agent can photoactivate drugs or otheragents in the medium about the composite.

Principle of Plasmonics-Enhancement Effect of the PEPST Probe UsingX-Ray Excitation

One embodiment of the basic PEPST probe embodiment comprises PAmolecules bound to an energy modulation agent and to plasmonic metal(gold) nanoparticles. First the metal nanoparticle can serve as a drugdelivery platform (see previous discussion). Secondly, the metalnanoparticle can play 2 roles:

(1) Enhancement of the X-ray electromagnetic field

(2) Enhancement of the emission signal of the energy modulation agentsystem.

The X ray radiation, used to excite the energy modulation agent system,is amplified by the metal nanoparticle due to plasmon resonance. As aresult the energy modulation agent system exhibits more emission lightthat is used to photoactivate the PA drug molecules (e.g., psoralens)and make them photoactive. In this case the metal nanoparticles aredesigned to exhibit strong plasmon resonance at or near the X raywavelengths. The surface plasmon resonance effect amplifies theexcitation light at the nanoparticles, resulting in increasedphotoactivation of the PA drug molecules and improved therapyefficiency. The plasmonics-enhanced mechanism can also be used with theother PEPST probes described above.

FIG. 18 illustrates the plasmonics-enhancement effect of the PEPSTprobe. X-ray used in medical diagnostic imaging has photon energies fromapproximately 10 to 150 keV, which is equivalent to wavelengths rangefrom 1.2 to 0.0083 Angstroms. [λ (Angstrom)=12.4/E (keV)]. Soft X raycan go to 10 nm. The dimension of plasmonics-active nanoparticlesusually have dimensions on the order or less than the wavelengths of theradiation used. Note that the approximate atomic radius of gold isapproximately 0.15 nanometers. At the limit, for gold the smallest“nanoparticle” size is 0.14 nm (only 1 gold atom). A nanoparticle withsize in the hundreds of nm will have approximately 10⁶-10⁷ gold atoms.Therefore, the range of gold nanoparticles discussed in this inventioncan range from 1-10⁷ gold atoms.

The gold nanoparticles can also enhance the energy modulation agentemission signal, which is use to excite the PA molecule. For psoralens,this spectral range is in the UVB region (320-400 nm). Silver or goldnanoparticles, nanoshell and nanocaps have been fabricated to exhibitstrong plasmon resonance in this region. FIG. 19 shows excitation andemission fluorescence spectra of a psoralen compound(8-methoxypsoralen).

PEPST Energy Modulation Agent-PA Probe with Detachable PA.

Some photoactive drugs require that the PA molecule to enter thenucleus. FIGS. 20A-20C show an embodiment of a PEPST probe where the PAdrug molecule is bound to the metal nanoparticles via a linker (FIG.20A) that can be cut by photon radiation (FIG. 20B). Such a probe isuseful for therapy modalities where the PA molecules have to enter thenucleus, e.g., psoralen molecules need to enter the nucleus of cells andintercalate onto DNA (FIG. 20C). Since it is more difficult for metalnanoparticles to enter the cell nucleus than for smaller molecules, itis preferable to use PEPST probes that have releasable PA molecules.

Suitable linkers for linking the PA drug molecule to the metalnanoparticles include, but are not limited to, labile chemical bondsthat can be broken by remote energy excitation (from outside the body,e.g., MW, IR, photoacoustic energy, ultrasound energy, etc.), labilechemical bonds that can be broken by the chemical environment insidecells, antibody-antigen, nucleic acid linkers, biotin-streptavidin, etc.

Nanoparticle Chain for Dual Plasmonics Effect

FIG. 21 illustrates an embodiment of the present invention PEPST probehaving a chain of metal particles having different sizes and coupled toeach other, which could exhibit such dual plasmonics-based enhancement.For example the parameters (size, metal type, structure, etc) of thelarger nanoparticle (FIG. 21 , left) can be tuned to NIR, VIS or UVlight while the smaller particle (FIG. 21 , right) can be tuned to Xray. There is also a coupling effect between these particles.

These nanoparticle chains are useful in providing plasmonics enhancementof both the incident radiation used (for example, x-ray activation ofCdS) as well as plasmonics enhancement of the emitted radiation thatwill then activate the PA.

Drug Delivery Platforms

Liposome Delivery of Energy Modulation Agent-PA Systems

The field of particle-based drug delivery is currently focused on twochemically distinct colloidal particles, liposomes and biodegradablepolymers. Both delivery systems encapsulate the active drug. The drug isreleased from the particle as it lyses, in the case of liposomes, ordisintegrates, as described for biodegradable polymers. One embodimentof the present invention uses liposomal delivery of energy modulationagent-PA systems (e.g., gold nanoshells) for therapy. An exemplaryembodiment is described below, but is not intended to be limiting to thespecific lipids, nanoparticles or other components recited, but ismerely for exemplary purposes:

Preparation of Liposomes. The liposome preparation method can be adaptedfrom Hölig et. al Hölig, P., Bach, M., Volkel, T., Nande, T., Hoffmann,S., Müller, R., and Kontermann, R. E., Novel RGD lipopeptides for thetargeting of liposomes to integrin-expressing endothelial and melanomacells. Protein Engineering Design and Selection, 2004. 17(5): p.433-441]. Briefly, the lipids PEG-DPPE, PC, and Rh-DPPE are mixed inchloroform in a round bottom flask and evaporated (Hieroglyph RotaryEvaporator, Rose Scientific Ltd., Edmonton, Alberta, Canada) toeliminate chloroform. The dry film is dehydrated into aqueous phase withusing PBS solution. A dry lipid film is prepared by rotary evaporationfrom a mixture of PC, cholesterol, and PEG-DPPE and then hydrated intoaqueous phase using PBS. The mixture is vigorously mixed by overtaxingand bath solicited (Instrument, Company) and the suspension extrudedthrough polycarbonate filter using Liposofast apparatus (Avestin Inc.,Ottawa, ON, Canada) (pore-size 0.8 μm). Preparation of liposomes isperformed as follows; 0.1 mmol of PC is dispersed in 8 ml of chloroformand supplemented with 0.5 mol of PEG-DPPE in 20 ml of chloroform. 0.3mmol rhodamine-labeled phosphatidylethanolamine (Rh-DPPE) is thenincorporated into the liposomes. The organic solvents are then removedby rotary evaporation at 35° C. for 2 h leaving a dry lipid film. Goldnanoshells are encapsulated into liposomes by adding them to the PBShydration buffer and successively into the dry lipid film. This mixtureis emulsified in a temperature controlled sonicator for 30 minutes at35° C. followed by vortexing for 5 min. Encapsulated gold nanoshells areseparated from unencapsulated gold nanoshells by centrifugation for 5minutes at 2400 r.p.m (1200 g). The resulting multilamellar vesiclessuspension is extruded through polycarbonate filter using Liposofastapparatus (Avestin Inc., Ottawa, ON, Canada) (pore-size 0.8 μm). Theaqueous mixture is obtained and stored at 4° C.

Fabrication of Gold Nanoparticles: The Frens method [Frens, G.,Controlled nucleation for the regulation of the particle size inmonodisperse gold solutions. Nature (London) Phys Sci, 1973. 241: p.20-22] can be used in the present invention to synthesize a solution ofgold nanoparticles ranging in diameter from 8-10 nm. Briefly, 5.0×10⁻⁶mol of HAuCl₄ is dissolved in 19 ml of deionized water producing a faintyellowish solution. This solution is heated with vigorous stirring in arotary evaporator for 45 minutes. 1 ml of 0.5% sodium citrate solutionis added and the solution is stirred for an additional 30 minutes. Thecolor of the solution gradually changed from the initial faint yellowishto clear, grey, purple and finally a tantalizing wine-red color similarto merlot. The sodium citrate used serves in a dual capacity, firstacting as a reducing agent, and second, producing negative citrate ionsthat are adsorbed onto the gold nanoparticles introducing surface chargethat repels the particles and preventing nanocluster formation.

Preparation and Internalization of Liposome-Encapsulated GoldNanoshells:

Liposome-encapsulated gold nanoshells are incubated with MCF-7 cellsgrown on partitioned cover-slips for intracellular delivery. This isdone by adding 10 μl of liposome-encapsulated gold nanoshells per 1 mlof cell culture medium. This is incubated for 30 minutes in a humidified(86% RH) incubator at 37° C. and 5% CO2. This cell is used forlocalization studies; to track the rhodamine-DPPE-labeled liposomes intothe cytoplasm of the MCF-7 cell. After incubation, the cells grown oncover-slips are washed three times in cold PBS and fixed using 3.7%formaldehyde in PBS. Rhodamine staining by rhodamine-DPPE-labeledliposomes is analyzed using a Nikon Diaphot 300 inverted microscope(Nikon, Inc., Melville, N.Y.).

Use of Ferritin and Apoferritin as Targeted Drug Delivery

Another embodiment to deliver the energy modulation agent-PA drugsinvolves the use of ferritin and apoferritin compounds. There isincreasing interest in ligand-receptor-mediated delivery systems due totheir non-immunogenic and site-specific targeting potential to theligand-specific bio-sites. Platinum anticancer drug have beenencapsulated in apoferritin. Ferritin, the principal iron storagemolecule in a wide variety of organisms, can also be used as a vehiclefor targeted drug delivery. It contains a hollow protein shell,apoferritin, which can contain up to its own weight of hydrous ferricoxide-phosphate as a microcrystalline micelle. The 24 subunits offerritin assemble automatically to form a hollow protein cage withinternal and external diameters of 8 and 12 nm, respectively. Eighthydrophilic channels of about 0.4 nm, formed at the intersections ofsubunits, penetrate the protein shell and lead to the protein cavity. Avariety of species such as gadolinium (Gd³⁺) contrast agents,desferrioxamine B, metal ions, and nanoparticles of iron salts can beaccommodated in the cage of apoferritin. Various metals such as iron,nickel, chromium and other materials have been incorporated intoapoferritin. Zinc selenide nanoparticles (ZnSe NPs) were synthesized inthe cavity of the cage-shaped protein apoferritin by designing a slowchemical reaction system, which employs tetraaminezinc ion andselenourea. The chemical synthesis of ZnSe NPs was realized in aspatially selective manner from an aqueous solution, and ZnSe cores wereformed in almost all apoferritin cavities with little bulkprecipitation.

A simple method for synthesizing gold nanoparticles stabilized by horsespleen apoferritin (HSAF) is reported using NaBH₄ or3-(N-morpholino)propanesulfonic acid (MOPS) as the reducing agent [LeiZhang, Joe Swift, Christopher A. Butts, Vijay Yerubandi and Ivan J.Dmochowski, Structure and activity of apoferritin-stabilized goldnanoparticles, Journal of Inorganic Biochemistry, Vol. 101, 1719-1729,2007]. Gold sulfite (Au₂S) nanoparticles were prepared in the cavity ofthe cage-shaped protein, apoferritin. Apoferritin has a cavity, 7 nm indiameter, and the diameter of fabricated Au₂S nanoparticles is about thesame size with the cavity and size dispersion was small. [KeikoYoshizawa, Kenji Iwahori, Kenji Sugimoto and Ichiro Yamashita,Fabrication of Gold Sulfide Nanoparticles Using the Protein Cage ofApoferritin, Chemistry Letters, Vol. 35 (2006), No. 10 p. 1192]. Thus,in one embodiment, the PA or energy modulation agent-PA compounds areencapsulated inside the apoferrtin shells.

Use of Ferritin and Apoferritin as Enhanced Targeting Agents

It was reported that ferritin could be internalized by some tumortissues, and the internalization was associated with themembrane-specific receptors. Previous studies have shown thatferritin-binding sites and the endocytosis of ferritin have beenidentified in neoplastic. Ferritin receptors have the potential for usein the delivery of anticancer drugs into the brain. In one embodiment,the present invention uses ferritin or apoferritin to both encapsulatePA and energy modulation agent-PA systems and also target tumor cellsselectively for enhanced drug delivery and subsequent phototherapy. Inthis case no additional bioreactors are needed.

FIGS. 22A-22D schematically illustrate the use of encapsulatedphotoactive agents (FIG. 22A) for delivery into tissue and subsequentrelease of the photoactive drugs after the encapsulated systems enterthe cell. Note the encapsulated system can have a bioreceptor forselective tumor targeting (FIG. 22B). Once inside the cell, the capsuleshell (e.g., liposomes, apoferritin, etc.) can be broken (FIG. 22C)using non-invasive excitation (e.g., ultrasound, RF, microwave, IR, etc)in order to release the photoactive molecules that can get into thenucleus and bind to DNA (FIG. 22D).

Non-Invasive Phototherapy Using PEPST Modality

FIGS. 23A-23B illustrate the basic operating principle of the PEPSTmodality. The PEPST photoactive drug molecules are given to a patient byoral ingestion, skin application, or by intravenous injection. The PEPSTdrugs travel through the blood stream inside the body towards thetargeted tumor (either via passive or active targeting strategies). Ifthe disease is systematic in nature a photon radiation at suitablewavelengths is used to irradiate the skin of the patient, the lightbeing selected to penetrate deep inside tissue (e.g., NIR or X ray). Forsolid tumors, the radiation light source is directed at the tumor.Subsequently a treatment procedure can be initiated using delivery ofenergy into the tumor site. One or several light sources may be used asdescribed in the previous sections. One embodiment of therapy comprisessending NIR radiation using an NIR laser through focusing optics.Focused beams of other radiation types, including but not limited to Xray, microwave, radio waves, etc. can also be used and will depend uponthe treatment modalities used.

Design, Fabrication and Operation of EIP Probes

FIGS. 25A-25B show various embodiments of the EIP probes:

-   -   (A) probe comprising PA molecules bound (through a linker, which        can be fixed or detachable) to an energy modulation agent        particle that can produce excitons under radiative excitation at        a suitable wavelength (e.g., X-ray). In this preferred        embodiment, the energy modulation agent materials have        structural defects that serve as traps for excitons.    -   (B) probe comprising PA molecules bound (through a linker, which        can be fixed or detachable) to an energy modulation agent        particle that can produce excitons under radiative excitation at        a suitable wavelength (e.g., X-ray). In this preferred        embodiment, the energy modulation agent materials have        impurities or dopant molecules that serve as traps for excitons.    -   (C)

EIP Probes with Tunable Emission:

The embodiment in probes B provide the capability to tune the energyconversion from an X ray excitation source into a wavelength of interestto excite the PA molecules. In 1976, D′Silva et al demonstrated thatpolynuclear aromatic hydrocarbons (PAH) molecules doped in a frozenn-alkane solids could be excited by X-ray and produce luminescence atvisible wavelengths characteristics of their luminescence spectra. [A.P. D'Silva, G. J. Oestreich, and V. A. Fassel, X-ray excited opticalluminescence of polynuclear aromatic hydrocarbons, Anal. Chem.; 1976;48(6) pp 915-917]. Tunable EIP probes can be designed to contain suchluminescent dopants such as highly luminescent PAHs exhibitingluminescence emission in the range of 300-400 nm suitable to activatepsoralen. A preferred embodiment of the EIP with tunable emissioncomprises a solid matrix (semiconductors, glass, quartz, conjugatedpolymers, etc) doped with naphthalene, phenanthrene, pyrene or othercompounds exhibiting luminescence (fluorescence) in the 300-400 nm range[T. Vo-Dinh, Multicomponent analysis by synchronous luminescencespectrometry, Anal. Chem.; 1978; 50(3) pp 396-401]. See FIGS. 26A-26E.The EEC matrix could be a semiconductor material, preferably transparentat optical wavelength of interest (excitation and emission).

Other dopant species such as rare earth materials can also be used asdopants. FIG. 27 shows the X ray excitation optical luminescence (XEOL)of Europium doped in a matrix of BaFBr, emitting at 370-420 nm. U.S.Patent Application Publication No. 2007/0063154 (hereby incorporated byreference) describes these and other nanocomposite materials (andmethods of making them) suitable for XEOL.

FIGS. 28A-28B show various embodiments of the EIP probes:

(A) probe comprising PA molecules bound around the energy modulationagent particle or embedded in a shell around an energy modulation agentparticle that can produce excitons under radiative excitation at asuitable wavelength (e.g., X-ray). In this preferred embodiment, theenergy modulation agent materials has structural defects that serve astraps for excitons.

(B) probe comprising PA molecules bound around the energy modulationagent particle or embedded in a shell around an energy modulation agentparticle that can produce excitons under radiative excitation at asuitable wavelength (e.g., X-ray). In this preferred embodiment, theenergy modulation agent materials have impurities or dopant moleculesthat serve as traps for excitons.

Design, Fabrication and Operation of EPEP Probes

FIGS. 29A-29B show various embodiments of EPEP probes of the presentinvention showing the exciton-plasmon coupling:

-   -   (A) probe comprising a PA molecule or group of PA molecules        bound (through a linker, which can be fixed or detachable) to an        energy modulation agent particle that can produce excitons under        radiative excitation at a suitable wavelength (e.g., X-ray). The        energy modulation agent particle is bound to (or in proximity        of) a metal nanoparticle covered with a nanoshell of silica (or        other dielectric material). The silica layer (or nanoshell) (see        FIG. 24A and FIG. 24B; layer nanoshell in white between energy        modulation material and metal nano structures) is designed to        prevent quenching of the luminescence light emitted by the        energy modulation agent particle excited by X-ray. The metal        nanoparticle (Au, Ag, etc) is designed to induce plasmons that        enhance the X ray excitation that subsequently leads to an        increase in the energy modulation agent light emission,        ultimately enhancing the efficiency of photoactivation, i.e.        phototherapy. The structure of the nanoparticle can also be        designed such that the plasmonics effect also enhances the        energy modulation agent emission light. These processes are due        to strong coupling between excitons (in the energy modulation        agent materials and plasmons in the metal nanoparticles; and    -   (B) probe comprising a PA molecule or group of PA molecules        bound (through a linker, which can be fixed or detachable) to an        energy modulation agent particle that can produce excitons under        radiative excitation at a suitable wavelength (e.g., X-ray). The        energy modulation agent particle is bound to (or in proximity        of) a metal nanoparticle via a spacer (linker). The spacer is        designed to prevent quenching of the luminescence light emitted        by the energy modulation agent particle excited by X-ray.

FIGS. 30A-30C show yet further embodiments of EPEP probes of the presentinvention:

(A) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is covered with a nanoshell of silica (or otherdielectric material), which is covered by a layer of separatenanostructures (nano islands, nanorods, nanocubes, etc. . . . ) of metal(Au, Ag). The silica layer (or other dielectric material) is designed toprevent quenching of the luminescence light emitted by the EEC (alsoreferred to as energy modulation agent) particle excited by X-ray. Themetal nanostructures (Au, Ag, etc) are designed to induce plasmons thatenhance the X ray excitation that subsequently leads to an increase inthe EEC light emission, ultimately enhancing the efficiency ofphotoactivation, i.e. phototherapy. The structure of the nanoparticlecan also be designed such that the plasmonics effect also enhance theenergy modulation agent emission light. These processes are due tostrong coupling between excitons (in the energy modulation agentmaterials and plasmons in the metal nanostructures).

(B) probe comprising a group of PA molecules in a particle bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The PA-containingparticle is covered with a layer of metallic nanostructures (Au, Ag).The metal nanostructures (Au, Ag, etc) are designed to induce plasmonsthat enhance the energy modulation agent light emission, ultimatelyenhancing the efficiency of photoactivation, i.e. phototherapy.

(C) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is covered with a nanoshell of silica (or otherdielectric material), which is covered by a layer of metallicnanostructures (Au, Ag). The silica layer (or other dielectric material)is designed to prevent quenching of the luminescence light emitted bythe energy modulation agent particle excited by X-ray. The metalnanostructures (Au, Ag, etc) are designed to induce plasmons thatenhance the X ray excitation that subsequently leads to an increase inthe energy modulation agent light emission, ultimately enhancing theefficiency of photoactivation, i.e. phototherapy. In addition. thePA-containing particle is covered with a layer of metallicnanostructures (Au, Ag). The metal nanostructures (Au, Ag, etc) aredesigned to induce plasmons that enhance the EEC light emission,ultimately enhancing the efficiency of photoactivation, i.e.phototherapy.

Hybrid EPEP Nano-Superstructures

EPEP probes can also comprise hybrid self-assembled superstructures madeof biological and abiotic nanoscale components, which can offerversatile molecular constructs with a spectrum of unique electronic,surface properties and photospectral properties for use in phototherapy.

FIGS. 31A-31B show various embodiments of EPEP probes of the presentinvention comprising superstructures of NPs, NWs and NRs:

(A) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanowire (ornanorod) covered with a nanoshell cylinder of silica (or otherdielectric material). The silica nanoshells cylinder is designed toprevent quenching of the luminescence light emitted by the energymodulation agent particle excited by X-ray. The metal nanoparticle (Au,Ag, etc) is designed to induce plasmons that enhance the X rayexcitation that subsequently leads to an increase in the energymodulation agent light emission, ultimately enhancing the efficiency ofphotoactivation, i.e. phototherapy. The structure of the nanoparticlecan also be designed such that the plasmonics effect and/or theexciton-plasmon coupling (EPC) effect also enhances the energymodulation agent emission light. These processes are due to strongcoupling between excitons (in the energy modulation agent materials andplasmons in the metal nanoparticles; and

(B) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanoparticlesvia a spacer (linker). The spacer is designed to prevent quenching ofthe luminescence light emitted by the energy modulation agent particleexcited by X-ray. Same effect as above in (A)

FIGS. 32A-32B show another set of embodiments of EPEP probes of thepresent invention comprising superstructures of NPs, NWs and NRs andbioreceptors (antibodies, DNA, surface cell receptors, etc.). The use ofbioreceptors to target tumor cells has been discussed above in relationto PEPST probes. Note that in this embodiment the PA molecules areattached along the NW axis in order to be excited by the emitting lightform the NWs.

FIG. 33 shows another embodiment of EPEP probes of the present inventioncomprising superstructures of NPs linked to multiple NWs.

For some embodiments, by adding metal nanostructures designed tointeract specifically with the excitons in the energy modulation agentsystem, there are significant improvements:

(1) an additional radiative pathway from exciton to photon conversion isintroduced

(2) the metal nanostructures can be designed to amplify (due to theplasmonics effect) the excitation radiation (e.g., X-ray) and/or theemission radiation (e.g, UV or visible) to excite the photo-active (PA)molecule, thereby enhancing the PA effectiveness.

Various metallic nanostructures that can be used in EPEP probeembodiments of the present invention are the same as those illustratedin FIG. 9 for the PEPST probes.

Experimental Methods

Preparation of Nanoparticles (Ag, Au)

There many methods to prepare metal nanoparticles for EPEP or PEPSTprobes. Procedures for preparing gold and silver colloids includeelectroexplosion, electrodeposition, gas phase condensation,electrochemical methods, and solution-phase chemical methods. Althoughthe methodologies for preparing homogeneous-sized spherical colloidalgold populations 2-40 nm in diameter are well known [N. R. Jana, L.Gearheart and C. J. Murphy, Seeding growth for size control of 5-40 nmdiameter gold nanoparticles. Langmuir 17 (2001), pp. 6782-6786], andparticles of this size are commercially available. An effective chemicalreduction method for preparing populations of silver particles (withhomogeneous optical scattering properties) or gold particles (withimproved control of size and shape monodispersity) is based on the useof small-diameter uniform-sized gold particles as nucleation centers forthe further growth of silver or gold layers.

One approach involves citrate reduction of a gold salt to produce 12-20nm size gold particles with a relatively narrow size distribution. Onemethod for producing smaller gold particles was developed by Brust et al[Brust, M.; Walker, M; Bethell, D.; Schiffiin, D. J; Whyman, R. Chem.Commun. 1994, 801]. This method is based on borohydride reduction ofgold salt in the presence of an alkanethiol capping agent to produce 1-3nmparticles. Nanoparticle sizes can be controlled between 2 and 5 nm byvarying the thiol concentration, [Hostetler, M. J.; Wingate, J. E.;Zhong, C. J.; Harris, J. E.; Vachet, R. W.; Clark, M R.; Londono, J. D.;Green, S. J.; Stokes, J. J; Wignall, G. D.; Glish, G. L.; Porter, M D.;Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 17]. Phosphine-stabilizedgold clusters have also been produced and subsequently converted tothiol-capped clusters by ligand exchange in order to improve theirstability [Schmid, G.; Pfeil, R.; Boese, R.; Bandrmann, F.; Meyer, S.;Calis, G. H. M.; van der Velden, J. W. A. Chem. Ber. 1981, 114, 3634;Warner, M. G.; Reed, S. M.; Hutchison, J. E. Chem. Mater. 2000, 12,3316.] and phosphine-stabilized monodispersed gold particles wereprepared using a similar protocol to the Brust method [Weare, W. W.;Reed, S. M.; Warner, M G.; Hutchison, J. E. J. Am. Chem. Soc. 2000, 122,12890]. See also recent review: Ziyi Zhong, Benoit¹ Male, Keith B.¹Luong, John H. T., More Recent Progress in the Preparation of AuNanostructures, Properties, and Applications, Analytical Letters; 2003,Vol. 36 Issue 15, p 3097-3118]

Fabrication of Nanoparticle of Metal coated with nanoshells of dyes

The Fabrication of Metal Nanoparticles Coated with Nanoshells of Dyemolecules can be performed using the method described by Masuhara et al[AKITO MASUHARA_, SATOSHI OHHASHly, HITOSHI KASAI; SHUJI OKADA,FABRICATION AND OPTICAL PROPERTIES OF NANOCOMPLEXES COMPOSED OF METALNANOPARTICLES AND ORGANIC DYES, Journal of Nonlinear Optical Physics &Materials Vol. 13, Nos. 3 & 4 (2004) 587-592]. Nanocomplexes composed ofAg or Au as a core and3-carboxlymethyl-5-[2-(3-octadecyl-2-benzoselenazolinylidene)ethylidene]rhodanine (MCSe) or copper (II) phthalocyanine (CuPc) as ashell are prepared by the co-reprecipitation method. In the case ofAg-MCSe nanocomplexes, 0.5 mM acetone solution of MCSe are injected into10 ml of Ag nanoparticle water dispersion, prepared by the reduction ofAgNO₃ using NaBH₄: Au-MCSe nanocomplexes are also fabricated in asimilar manner. A water dispersion of Au nanoparticles was prepared bythe reduction of HAuCl₄ using sodium citrate. Subsequently, 2 M NH₄OH(50 μl) was added and the mixture was thermally treated at 50° C. Thisamine treatment often stimulates the J-aggregate formation of MCSe.6Ag-CuPc and Au-CuPc nanocomplexes were also fabricated in the samemanner: 1 mM 1-methyl-2-pyrrolidinone (NMP) solution of CuPc (200 μl)was injected into a water dispersion (10 ml) of Ag or Au nanoparticles.

The present invention treatment may also be used for inducing an autovaccine effect for malignant cells, including those in solid tumors. Tothe extent that any rapidly dividing cells or stem cells may be damagedby a systemic treatment, then it may be preferable to direct thestimulating energy directly toward the tumor, preventing damage to mostnormal, healthy cells or stem cells by avoiding photo activation orresonant energy transfer of the photoactivatable agent.

Alternatively, a treatment may be applied that slows or pauses mitosis.Such a treatment is capable of slowing the division of rapidly dividinghealthy cells or stem cells during the treatment, without pausingmitosis of cancerous cells. Alternatively, a blocking agent isadministered preferentially to malignant cells prior to administeringthe treatment that slows mitosis.

In one embodiment, an aggressive cell proliferation disorder has a muchhigher rate of mitosis, which leads to selective destruction of adisproportionate share of the malignant cells during even a systemicallyadministered treatment. Stem cells and healthy cells may be spared fromwholesale programmed cell death, even if exposed to photoactivatedagents, provided that such photoactivated agents degenerate from theexcited state to a lower energy state prior to binding, mitosis or othermechanisms for creating damage to the cells of a substantial fraction ofthe healthy stem cells. Thus, an auto-immune response may not beinduced.

Alternatively, a blocking agent may be used that prevents or reducesdamage to stem cells or healthy cells, selectively, which wouldotherwise be impaired. The blocking agent is selected or is administeredsuch that the blocking agent does not impart a similar benefit tomalignant cells, for example.

In one embodiment, stem cells are targeted, specifically, fordestruction with the intention of replacing the stem cells with a donorcell line or previously stored, healthy cells of the patient. In thiscase, no blocking agent is used. Instead, a carrier or photosensitizeris used that specifically targets the stem cells.

In a further embodiment, methods in accordance with the presentinvention may further include adding an additive to alleviate treatmentside-effects. Exemplary additives may include, but are not limited to,antioxidants, adjuvant, or combinations thereof. In one exemplaryembodiment, psoralen is used as the activatable pharmaceutical agent,UV-A is used as the activating energy, and antioxidants are added toreduce the unwanted side-effects of irradiation.

An advantage of the methods of the present invention is that byspecifically targeting cells affected by a cell proliferation disorder,such as rapidly dividing cells, and triggering a cellular change, suchas apoptosis, in these cells in situ, the immune system of the host maybe stimulated to have an immune response against the diseased cells.Once the host's own immune system is stimulated to have such a response,other diseased cells that are not treated by the activatablepharmaceutical agent may be recognized and be destroyed by the host'sown immune system. Such autovaccine effects may be obtained, forexample, in treatments using psoralen and UV-A.

The present invention methods can be used alone or in combination withother therapies for treatment of cell proliferation disorders.Additionally, the present invention methods can be used, if desired, inconjunction with recent advances in chronomedicine, such as thatdetailed in Giacchetti et al, Journal of Clinical Oncology, Vol 24, No22 (August 1), 2006: pp. 3562-3569. In chronomedicine it has been foundthat cells suffering from certain types of disorders, such as cancer,respond better at certain times of the day than at others. Thus,chronomedicine could be used in conjunction with the present methods inorder to augment the effect of the treatments of the present invention.

In another aspect, the present invention further provides systems andkits for practicing the above described methods.

In one embodiment, a system in accordance with the present invention mayinclude: (1) an initiation energy source; (2) one or more energymodulation agents; and (3) one or more activatable pharmaceuticalagents.

In another embodiment, a system in accordance with the present inventionmay include an initiation energy source and one or more activatablepharmaceutical agents.

In preferred embodiments, the initiation energy source may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. One example of such linear accelerators is the SmartBeam™IMRT (intensity modulated radiation therapy) system from Varian medicalsystems (Varian Medical Systems, Inc., Palo Alto, Calif.).

In other embodiments, endoscopic or laproscopic devices equipped withappropriate initiation energy emitter may be used as the initiationenergy source. In such systems, the initiation energy may be navigatedand positioned at the pre-selected coordinate to deliver the desiredamount of initiation energy to the site. In other embodiments,endoscopic or laproscopic devices equipped with appropriate energymodulation agents (encapsulated from the biological medium) may be usedto provide different wavelengths of UV or visible or infraredirradiation in a vicinity of or within a target structure.

In further embodiments, dose calculation and robotic manipulationdevices may also be included in the system.

The reagents and chemicals useful for methods and systems of the presentinvention may be packaged in kits to facilitate application of thepresent invention. Such a kit for modifying a target structure wouldcomprise at least one agent selected from the group consisting of energymodulation agents, plasmonics-active agents and combinations thereof.The energy modulation agents would be one or more light emitters capableof emitting at least two different wavelengths of light, each wavelengthof light associated with a different biological response, and thedifferent wavelengths capable of activating different biologicalresponses. The kit would preferably include one or more containerssuitable for storing the agents in stable forms.

In one exemplary embodiment, a kit including a psoralen, andfractionating containers for easy fractionation and isolation ofautovaccines is contemplated. A further embodiment of kit would compriseat least one activatable pharmaceutical agent capable of causing apredetermined cellular change, at least one energy modulation agentcapable of activating the at least one activatable agent when energized,at least one plasmonics agent and containers suitable for storing theagents in stable form, and preferably further comprising instructionsfor administering the at least one activatable pharmaceutical agent, atleast one plasmonics agent and at least one energy modulation agent to asubject, and for applying an initiation energy from an initiation energysource to activate the activatable pharmaceutical agent. Theinstructions could be in any desired form, including but not limited to,printed on a kit insert, printed on one or more containers, as well aselectronically stored instructions provided on an electronic storagemedium, such as a computer readable storage medium. Also optionallyincluded is a software package on a computer readable storage mediumthat permits the user to integrate the information and calculate acontrol dose, to calculate and control intensity of the irradiationsource.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES

Preparation of Silver Nanoparticles

Silver (or gold) colloids were prepared according to the standardLee-Meisel method: 200 mL of 10⁻³ M AgNO₃ aqueous solution was boiledunder vigorous stirring, then 5 mL of 35-mM sodium citrate solution wereadded and the resulting mixture was kept boiling for 1 h. This procedurewas reported to yield ˜10¹¹ particles/mL of homogenously sized colloidalparticles with a diameter of ˜35-50 nm and an absorption maximum at 390nm. The colloidal solutions were stored at 4° C. and protected from roomlight. Further dilutions of the colloidal solutions were carried outusing distilled water.

Fabrication/Preparation of Metal Nanocaps

One approach has involved the use of nanospheres spin-coated on a solidsupport in order to produce and control the desired roughness. Thenanostructured support is subsequently covered with a layer of silverthat provides the conduction electrons required for the surface plasmonmechanisms. Among the techniques based on solid substrates, the methodsusing simple nanomaterials, such as Teflon or latex nanospheres, appearto be the simplest to prepare. Teflon and latex nanospheres arecommercially available in a wide variety of sizes. The shapes of thesematerials are very regular and their size can be selected for optimalenhancement. These materials comprise isolated dielectric nanospheres(30-nm diameter) coated with silver producing systems ofhalf-nanoshells, referred to as nanocaps.

FIG. 24 shows a scanning electron micrograph (SEM) of 300-nm diameterpolymer nanospheres covered by a 100-nm thick silver nanocaps(half-nanoshell) coating. The nanoparticles can be sonicated to releasethem from the underlying substrate. The effect of the sphere size andmetal layer thickness upon the SERS effect can be easily investigated.The silver coated nanospheres were found to be among the mostplasmonics-active investigated. Gold can also be used instead of silverto coat over nanoparticles comprising PA drug molecules.

Fabrication of Gold Nanoshells

Gold nanoshells have been prepared using the method described by Hirschet al. [Hirsch L R, Stafford R J, Bankson J A, Sershen S R, Price R E,Hazle J D, Halas N J, West J L (2003) Nanoshell-mediated near infraredthermal therapy of tumors under MR Guidance. Proc Natl Acad Sci100:13549-13554] using a mechanism involving nucleation and thensuccessive growth of gold nanoparticles around a silica dielectric core.Gold nanoparticles, the seed, prepared as described above using theFrens method, were used to grow the gold shell. Silica nanoparticles(100 nm) used for the core of the nanoshells were monodispersed insolution of 1% APTES in EtOH. The gold “seed” colloid synthesized usingthe Frens method were grown onto the surface of silica nanoparticles viamolecular linkage of amine groups. The “seed” covers the aminated silicananoparticle surface, first as a discontinuous gold metal layergradually growing forming a continuous gold shell. Gold nanoparticlesused as the “seed” were characterized using optical transmissionspectroscopy (UV-Vis Spectrophotometer, Beckman Coulter, Fullerton,Calif.) and atomic force microscopy (Atomic Force Microscope, VeecoInstruments, Woodbury, N.Y.) while gold nanoshells were characterizedusing optical transmission spectroscopy and scanning electron microscopy(Scanning Electron Microscope, Hitachi S-4700, Hitachi High TechnologiesAmerica, Inc. Pleasanton, N.Y.).

Non-Medical Applications

Referring to FIG. 3-1 , an exemplary system according to one embodimentof the invention may have an initiation energy source 1 directed at ageneric medium 4 (biological or non-biological). Activatable agents 2and an energy modulation agents 3 (such as the diamond and DLC-basedenergy modulation agents and/or the other energy modulation agentsdescribed above) are dispersed throughout the medium 4. The initiationenergy source 1 may additionally be connected via a network 8 to acomputer system 5 capable of directing the delivery of the initiationenergy. In various embodiments, the energy modulation agents 3 areencapsulated energy modulation agents 6, depicted in FIG. 3-1 as silicaencased energy modulation agents. As shown in FIG. 3-1 , initiationenergy 7 in the form of radiation from the initiation energy source 1permeated throughout the medium 4. A more thorough discussion of thecomputer system 5 is provided below in reference to FIGS. 4 and 5 . Theinitiation energy source 1 can be an external energy source or an energysource located at least partially in the medium 4. Activatable agents 2and/or the energy modulation agents 3 can include plasmonics agentswhich enhance either the applied energy or the energy emitted from theenergy modulation agents 3 so as to directly or indirectly produce achange in the medium.

In various embodiments, the initiation energy source 1 may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. One example of such linear accelerators is the SmartBeam™IMRT (intensity modulated radiation therapy) system from Varian medicalsystems (Varian Medical Systems, Inc., Palo Alto, Calif.). In otherembodiments, the initiation energy source 1 may be commerciallyavailable components of X-ray machines or non-medical X-ray machines.X-ray machines that produce from 10 to 150 keV X-rays are readilyavailable in the marketplace. For instance, the General ElectricDefinium series or the Siemens MULTIX series are but two examples oftypical X-ray machines designed for the medical industry, while theEagle Pack series from Smith Detection is an example of a non-medicalX-ray machine. As such, the invention is capable of performing itsdesired function when used in conjunction with commercial X-rayequipment.

In other embodiments, the initiation energy source 1 can be a radiofrequency or microwave source emitting radio waves at a frequency whichpermeates the medium and which triggers or produces secondary radiantenergy emission within the medium by interaction with the energymodulation elements 6 therein. In other embodiments, the initiationenergy source 1 can be an ultraviolet, visible, near infrared (NIR) orinfrared (IR) emitter emitting at a frequency which permeates the medium4 and which triggers or produces secondary radiant energy emissionwithin medium 4 by interaction with the energy modulation elements 6therein.

The energy modulation structures of this invention can be provided onthe interior of sealed quartz or glass tubes or can be provided coatedon the surface of spheres or tubes, and further encapsulated with asilicate or another passivation layer. It is known that ultraviolet (UV)with a wavelength of 254 nm tends to inactivate most types ofmicroorganisms. The deep UV diamond emission lines shown in FIGS. 34-1and 34-2 make diamond (and DLC) a suitable choice for inclusion with theother energy modulation structures described herein or as a sole part ofthe energy modulation structures of this invention.

As on example of a non-medical application, most juices are opaque to UVdue to the high-suspended solids in them and hence the conventional UVtreatment, usually used for water treatment, cannot be used for treatingjuices. In order to make the process efficient, a thin film reactorconstructed from glass has been used with the juice flowing along theinner surface of a vertical glass tube as a thin film. See “UltravioletTreatment of Orange Juice” by Tran et al. published in Innovative FoodScience & Emerging Technologies (Volume 5, Issue 4, December 2004, Pages495-502), the entire contents of which are incorporated herein byreference. Tran et al. reported that decimal reduction doses requiredfor the reconstitute orange juices (OJ; 10.5° Brix) were 87±7 and 119±17mJ/cm² for the standard aerobic plate count (APC) and yeast and moulds,respectively. They also reported that the shelf life of fresh squeezedorange juice was extended to 5 days with a limited exposure of UV (73.8mJ/cm²). The effect of UV on the concentration of Vitamin C wasinvestigated using both HPLC and titration methods of measurements. Thedegradation of Vitamin C was 17% under high UV exposure of 100 mJ/cm²,which was similar to that usually found in thermal sterilization. Enzymepectin methylesterase (PME) activity, which is the major cause of cloudloss of juices, was also measured. The energy required for UV treatmentof orange juice (2.0 kW h/m³) was much smaller than that required inthermal treatment (82 kW h/m³). The color and pH of the juice were notsignificantly influenced by the treatment.

The invention described herein offers advantages over this approach inthat the energy modulation structures of this invention (including thediamond or DLC materials) can be placed inside fixtures such as quartzor glass (encapsulation structures) within the orange juice (or otherfluid medium) and irradiated with x-ray or other high energy sourcesupplied for example to the container to activate the encapsulatedstructures of the invention in the orange juice.

While discussed with regard to orange juice, any other medium to besterilized including food products, medical products and cosmeticproducts could be treated using the technique of the invention describedherein.

In one embodiment, the energy modulation structures of this invention(including the diamond or DLC materials) are complexed with other X-raydown converting particles or other energy modulation agents permittingfor example X-ray irradiation to also assist in this process.

Sterilization of Blood Products

U.S. Pat. No. 6,087,141 (the entire contents of which are incorporatedherein by reference) describes an ultraviolet light activated psoralenprocess for sterilization of blood transfusion products. The presentinvention can be applied for the neutralization of AIDS and HIV or otherviral or pathogenic agents in blood transfusion products. In thisembodiment, at least one photoactivatable agent is selected frompsoralens, pyrene cholesteryloleate, acridine, porphyrin, fluorescein,rhodamine, 16-diazorcortisone, ethidium, transition metal complexes ofbleomycin, transition metal complexes of deglycobleomycin organoplatinumcomplexes, alloxazines, vitamin Ks, vitamin L, vitamin metabolites,vitamin precursors, naphthoquinones, naphthalenes, naphthols andderivatives thereof having planar molecular conformations,porphorinporphyrins, dyes and phenothiazine derivatives, coumarins,quinolones, quinones, anthroquinones, porphycene, rubyrin, rosarin,hexaphyrin, sapphyrin, chlorophyl, chlorin, phthalocynine, porphyrazine,bacteriochlorophyl, pheophytin, texaphyrin macrocyclic-based component,or a metalated derivative thereof. These photoactivatable agents serveas recipients for the secondarily generated light induced by the downconversion or upconversion.

In various embodiments of the invention, the UV or visible lightrecipients are secondary agents performing other functions. Suitablesecondary agents for the invention include secondary emitters, cytotoxicagents, magnetic resonance imaging (MRI) agents, positron emissiontomography (PET) agents, radiological imaging agents, or photodynamictherapy (PDT) agents.

These photoactivatable agents (recipients and secondary agents) areintroduced into the blood product (or a patient's blood stream). Aninitiation energy such as x-ray (or other high energy source, deep UV,electrons, gamma rays) is applied to the blood product (dr to thepatient). The energy modulation structures of this invention (includingthe diamond or DLC materials) invention (either included in the bloodproduct or in encapsulated structures) generate secondary light such asUV or visible light which activates the photoactivatable agents in theblood products.

In a specific example, the photoactivatable agent is a psoralen, acoumarin, or a derivative thereof, and as discussed above, one cansterilize blood products in vivo (i.e., in a patient) or in a containerof the blood product (such as for example donated blood). The treatmentcan be applied to treat disorders such as for example a cancer cell, atumor cell, an autoimmune deficiency symptom virus, or a blood-bornegermicide is treated by the psoralen, the coumarin, or the derivativethereof.

Waste Water Detoxification

Photocatalysis has also been used as tertiary treatment for wastewaterto comply with regulatory discharge limits and to oxidize compounds thathave not been oxidized in the biological treatment. Photocatalysis hasbeen used to reduce or eliminate several pollutants (e.g., alkanes,alkenes, phenols, aromatics, pesticides) with great success. In manycases, total mineralization of the organic compounds has been observed.Several photocatalysts, such as CdS, Fe₂O₃, ZnO, WO₃, and ZnS, have beenstudied, but the best results have been achieved with TiO₂P₂₅. Thesephotocatalyst can be used in the invention.

The wastewaters of an oil refinery are the waters resulting from washingthe equipment used in the process, undesirable wastes, and sanitarysewage. These effluents have high oil and grease contents, besides otherorganic compounds in solution. These pollutants form a residual chemicaloxygen demand (COD) that may pose serious toxic hazards to theenvironment.

It is known that photocatalysis can be used for waste water reductionremediation. U.S. Pat. No. 5,118,422 (the entire contents of which areincorporated herein by reference) to Cooper et al. describe anultraviolet driven photocatalytic post-treatment technique for purifyinga water feedstock containing an oxidizable contaminant compound. In thiswork, the water feedstock was mixed with photocatalytic semiconductorparticles (e.g., TiO₂, ZnO, CdS, CdSe, SnO₂, SrTiO₃, WO₃, Fe₂O₃, andTa₂O₅ particles) having a particle size in the range of about 0.01 toabout 1.0 micron and in an amount of between about 0.01% and about 0.2%by weight of the water. The water including the semiconductor mixture isexposed to band-gap photons for a time sufficient to affect an oxidationof the oxidizable contaminant to purify the water. Crossflow membranefiltration was used to separate the purified water from thesemiconductor particles. Cooper et al. show that the organic impuritycarbon content of simulated reclamation waters at nominal 40 PPM levelwere reduced to parts per billion using a recirculation batch reactor.Cooper et al. identified that one important aspect of the photocatalyticprocess is the adsorption of the organic molecules onto the extremelylarge surface area presented by the finely divided powders dispersed inthe water. Cooper et al. further indicated that, in photoelectrochemicalapplications, advantage is taken of the fact that the solid phase (ametal oxide semiconductor) is also photo-active and that the generatedcharge carriers are directly involved in the organic oxidation. Theadsorption of the band-gap photon by the semiconductor particle resultsin the formation of an electron (e⁻)/hole (h⁺) pair. Cooper et al.explain that the electrons generated in the conduction band react withsolution oxygen forming the dioxygen anion (O₂₋) species whichsubsequently undergo further reactions resulting in the production ofthe powerfully oxidizing hydroxyl radical species, ⁻OH. These powerfuloxidants are known to oxidize organic compounds by themselves.Additionally, Cooper et al. explain that the strongly oxidizing holesgenerated in the valence band have sufficient energy to oxidize allorganic bonds.

In the reactor of Cooper et al., turbulence is necessary in order toensure that the waste water contaminants and the photocatalytic titaniaparticles are exposed to the UV light. Cooper et al. explain that themost basic considerations of photocatalyst light adsorption and itsrelationship to convective mixing. For a 0.1 wt % photocatalyst loading,experiments have shown that 90% of the light is absorbed within 0.08 cm.This is primarily due to the large UV absorption coefficient of thephotocatalyst and therefore, most of the photoelectrochemistry occurswithin this illuminated region. By operating the reactor of Cooper etal. with a Reynolds number (Re) of 4000, a significant portion of thephotoactive region is ensured of being within the well mixed turbulentzone.

Santos et al. have reported in “Photocatalysis as a tertiary treatmentfor petroleum refinery wastewaters” published in Braz. J. Chem. Eng.vol. 23, No. 4, 2006 (the entire contents of which are incorporatedherein by reference), photocatalysis for tertiary treatment forpetroleum refinery wastewaters which satisfactorily reduced the amountof pollutants to the level of the regulatory discharge limits andoxidized persistent compounds that had not been oxidized in thebiological treatment. The treatment sequence used by the refinery(REDUC/PETROBRAS, a Brazilian oil refinery) is oil/water separationfollowed by a biological treatment. Although the process efficiency interms of biological oxygen demand (BOD) removal is high, a residual andpersistent COD and a phenol content remains. The refining capacity ofthe refinery is 41,000 m³/day, generating 1,100 m³/h of wastewater,which are discharged directly into the Guanabara Bay (Rio de Janeiro).Treating the residual and persistent COD remains a priority.

Santos et al. conducted a first set of experiments carried out in anopen 250 mL reactor containing 60 mL of wastewater. In the second set ofexperiments, a Pyrex® annular reactor containing 550 mL of wastewaterwas used (De Paoli and Rodrigues, 1978), as shown in FIG. 1 . Thereaction mixtures inside the reactors were maintained in suspension bymagnetic stirring. In all experiments, air was continuously bubbledthrough the suspensions. A 250 W Phillips HPL-medium pressure mercuryvapor lamp (with its outer bulb removed) was used as the UV-light source(radiant flux of 108 J·m⁻²·s⁻¹ at λ>254 nm). In one set of experiments,the lamp was positioned above the surface of the liquid at a fixedheight (12 cm). In the second set, the lamp was inserted into the well.All experiments by Santos et al. were performed at 25±1° C. The catalystconcentration ranged from 0.5 to 5.5 g L⁻¹ and the initial pH rangedfrom 3.5 to 9.

In one embodiment of the invention described herein, the energymodulation structures of this invention (including the diamond or DLCmaterials) would be placed inside quartz or glass fixtures within thewaste water or would be placed on silica encapsulated structures withinthe waste water which, like the photocatalytic TiO₂, could be entrainedin the waste water during the irradiation.

Upon x-ray or other high energy radiation (or other high energy source,deep UV, electrons, gamma rays), the energy modulation structures ofthis invention (including the diamond or DLC materials) would generateUV light in nearby presence of the photocatalytic agent. In other wordsfor this embodiment, the energy modulation structures of this invention(including the diamond or DLC materials) are mixed along with thephotocatalytic semiconductor particles in the waste water fluid stream,and the exterior activation energy source penetrates the container(e.g., a plastic or aluminum container) and irradiates the bulk of thewaste water, producing UV light throughout the waste water which in turndrives the photocatalytic reactions.

Photostimulation

Photostimulation is a field in which light is applied to in order toalter or change a physical property. For example, there has been anincreased focus on the use of biodegradable polymers in consumer andbiomedical fields. Polylactic acid (PLA) plastics andpolyhydroxyalkanoates (PHA) plastics have been playing a vital role infulfilling the objectives. But their relatively hydrophobic surfaceslimit their use in various applications. Hence, there is a need tosurface modify these film surfaces. Due to the lack of any modifiableside chain groups, workers have used a sequential two step photograftingtechnique for the surface modification of these biopolymers. In stepone, benzophenone was photografted on the film surface and in step two,hydrophilic monomers like acrylic acid and acrylamide werephotopolymerized from the film surfaces.

Workers have found that UV irradiation could realize an effective graftcopolymerization. UV-assisted photografting in ethanol has been used togrow hydrophilic polymers (e.g., poly(acrylic acid) and polyacrylamide)from the surfaces of PLA, PHA, and PLA/PHA blend films. In that work, afunctional polyurethane (PU) surface was prepared by photo-graftingN,N-dimethylaminoethyl methacrylate (DMAEM) onto the membrane surface.Grafting copolymerization was conducted by the combined use of thephoto-oxidation and irradiation grafting. PU membrane was photo-oxidizedto introduce the hydroperoxide groups onto the surface, then themembrane previously immersed in monomer solution was irradiated by UVlight. Results have shown prior to the invention that UV irradiation canrealize graft copolymerization effectively.

In the invention described herein, these processes are expedited by theinclusion of the energy modulation structures of this invention(including the diamond or DLC materials) in dispersion in the fluidmedium being used for photostimulation. Upon irradiation, thesestructures would generate UV light within the NIR penetration depth ofthe medium and permitting batch or bulk type processing to occur inparallel inside the container. Further, when laser light is used for theNIR, the plastic surface can be “written” onto such that inks wouldselectively absorb on those regions where surface of the polymer wasexposed to the UV generated light.

Photodeactivation

In many industrial processes, especially food and beverage industries,yeasts are used to produce changes in a medium such as the conversion ofsugars in the raw product. One particularly prominent example is in thewine industry. Stopping the wine from fermenting any further wouldpreserve the current level of sweetness. Likewise, allowing the wine tocontinue fermenting further would only make the wine less sweet witheach passing day. Eventually the wine would become completely dry atwhich time the fermentation would stop on its own. This is becauseduring the fermentation process yeast turns the sugar into alcohol.

Wanting to stop the fermentation process is all good in and of itself.But unfortunately, there is really no practical way to successfully stopa fermentation dead in its tracks. Additives such as sulphite andsorbate can be added to stabilize a fermented product and stopadditional fermentation. Many winemakers will turn to sulfites such asthat found in Sodium Bisulfite or Campden tablets for the answer. But,these two items are not capable of reliably killing enough of the yeastto guarantee a complete stop of the activity—at least not at normaldoses that leave the wine still drinkable.

Once the bulk of the sulfites from either of these ingredients dissipatefrom the wine into the air—as sulfites do—there is a very strong chancethat the remaining few live yeast cells will start multiplying andfermenting again if given enough time. This usually happens at a mostinconvenient time, like after the wine has been bottled and stowed away.

Potassium sorbate is another ingredient that many winemakers considerwhen trying to stop a wine from fermenting any further. There is a lotof misunderstanding surrounding this product. It is typically called forby home wine making books when sweetening a wine. This is a situationwhere the fermentation has already completed and is ready for bottling.One adds the potassium sorbate along with the sugar that is added forsweetening.

The potassium sorbate stops the yeast from fermenting the newly addedsugar. So, many winemakers assume potassium sorbate can stop an activefermentation as well, but, potassium sorbate does not kill the yeast atall, but rather it makes the yeast sterile. In other words, it impairsthe yeast's ability to reproduce itself. But, it does not hinder theyeast's ability to ferment sugar into alcohol.

Ultraviolet light is known to destroy yeast cultures, but has restrictedapplications due to the inability of UV light to penetrate throughoutthe fluid medium. While heat can be used to destroy the yeast activity,cooking of the product may be premature or may produce undesirablechanges in the consistency and taste. For liquid or fluid food products,the same techniques described above could be used for the applicationdescribed here. For non-liquid products, energy modulation agents withlittle and preferably no toxicity (e.g. Fe oxides or titanium oxides)could be added. Here, the concentration of these additives would likelybe limited by any unexpected changes in taste.

In one embodiment, the energy modulation structures of this invention(including the diamond or DLC materials) are included in the yeastcontaining media, and with for example X-ray irradiation (or other highenergy source, deep UV, electrons, gamma rays) stops the fermentingprocess.

Photoactivated Cross-Linking and Curing of Polymers

In this application, the energy modulation structures of this invention(including the diamond or DLC materials) are provided and distributedinto an uncured polymer based medium for the activation ofphotosensitive agents in the medium to promote cross-linking and curingof the polymer based medium. In one embodiment, the diamond or DLCmaterials structures of the invention are complexed with otherdown-converting luminescent particles or other energy modulation agentsprior to being added to the polymer.

For adhesive and surface coating applications, light activatedprocessing is limited due to the penetration depth of UV light into theprocessed medium. In light activated adhesive and surface coatingprocessing, the primary limitation is that the material to be cured mustsee the light—both in type (wavelength or spectral distribution) andintensity. This limitation has meant that one medium typically has totransmit the appropriate light. In adhesive and surface coatingapplications, any “shaded” area will require a secondary cure mechanism,increasing cure time over the non-shaded areas and further delaying curetime due to the existent of a sealed skin through which subsequentcuring must proceed.

Conventionally, moisture-curing mechanisms, heat-curing mechanisms, andphoto-initiated curing mechanisms are used to initiate cure, i.e.,cross-linking, of reactive compositions, such as reactive silicones,polymers, and adhesives. These mechanisms are based on eithercondensation reactions, whereby moisture hydrolyzes certain groups, oraddition reactions that can be initiated by a form of energy, such aselectromagnetic radiation or heat.

The invention described herein can use any of the following lightactivated curing polymers as well as others known in the art to whichthe upconverter structures of the invention are added.

For example, one suitable light activated polymer compound includes UVcuring silicones having methacrylate functional groups. U.S. Pat. No.4,675,346 to Lin, the disclosure of which is hereby expresslyincorporated herein by reference, is directed to UV curable siliconecompositions including at least 50% of a specific type of siliconeresin, at least 10% of a fumed silica filler and a photoinitiator, andcured compositions thereof. Other known UV curing silicone compositionssuitable for the invention include organopolysiloxane containing a(meth)acrylate functional group, a photosensitizer, and a solvent, whichcures to a hard film. Other known UV curing silicone compositionssuitable for the invention include compositions of an organopolysiloxanehaving an average of at least one acryloxy and/or methacryloxy group permolecule; a low molecular weight polyacrylyl crosslinking agent; and aphotosensitizer.

Loctite Corporation has designed and developed UV and UV/moisture dualcurable silicone compositions, which also demonstrate high resistance toflammability and combustibility, where the flame-retardant component isa combination of hydrated alumina and a member selected from the groupconsisting of organo ligand complexes of transition metals,organosiloxane ligand complexes of transition metals, and combinationsthereof. See U.S. Pat. Nos. 6,281,261 and 6,323,253 to Bennington. Theseformulations are also suitable for the invention.

Other known UV photoactivatable silicones include siliconesfunctionalized with, for example, carboxylate, maleate, cinnamate andcombinations thereof. These formulations are also suitable for theinvention. Other known UV photoactivatable silicones suitable for theinvention include benzoin ethers (“UV free radical generator”) and afree-radical polymerizable functional silicone polymers, as described inU.S. Pat. No. 6,051,625 whose content is incorporated herein byreference in its entirety. The UV free radical generator (i.e., thebenzoin ether) is contained at from 0.001 to 10 wt % based on the totalweight of the curable composition. Free radicals produced by irradiatingthe composition function as initiators of the polymerization reaction,and the free radical generator can be added in a catalytic quantityrelative to the polymerizable functionality in the subject composition.Further included in these silicone resins can be silicon-bonded divalentoxygen atom compounds which can form a siloxane bond while the remainingoxygen in each case can be bonded to another silicon to form a siloxanebond, or can be bonded to methyl or ethyl to form an alkoxy group, orcan be bonded to hydrogen to form silanol. Such compounds can includetrimethylsilyl, dimethylsilyl, phenyldimethylsilyl, vinyldimethylsilyl,trifluoropropyldimethylsilyl, (4-vinylphenyl)dimethylsilyl,(vinylbenzyl)dimethylsilyl, and (vinylphenethyl)dimethylsilyl.

The photoinitiator component of the invention is not limited to thosefree radical generators given above, but may be any photoinitiator knownin the art, including the afore-mentioned benzoin and substitutedbenzoins (such as alkyl ester substituted benzoins), Michler's ketone,dialkoxyacetophenones, such as diethoxyacetophenone (“DEAP”),benzophenone and substituted benzophenones, acetophenone and substitutedacetophenones, and xanthone and substituted xanthones. Other desirablephotoinitiators include DEAP, benzoin methyl ether, benzoin ethyl ether,benzoin isopropyl ether, diethoxyxanthone, chloro-thio-xanthone,azo-bisisobutyronitrile, N-methyl diethanolaminebenzophenone, andmixtures thereof. Visible light initiators include camphoquinone,peroxyester initiators and non-fluorene-carboxylic acid peroxyesters.

Commercially available examples of photoinitiators suitable for theinvention include those from Vantico, Inc., Brewster, N.Y. under theIRGACURE and DAROCUR tradenames, specifically IRGACURE 184(1-hydroxycyclohexyl phenyl ketone), 907(2-methyl-1-[4-(methylthio)phenyl]-2-morpholino propan-1-one), 369(2-benzyl-2-N,N-dimethylamino-1-(4-morpholinophenyl)-1-butanone), 500(the combination of 1-hydroxy cyclohexyl phenyl ketone andbenzophenone), 651 (2,2-dimethoxy-2-phenyl acetophenone), 1700 (thecombination of bis(2,6-dimethoxybenzoyl-2,4,4-trimethyl pentyl)phosphine oxide and 2-hydroxy-2-methyl-1-phenyl-propan-1-one), and 819[bis(2,4,6-trimethyl benzoyl)phenyl phosphine oxide] and DAROCUR 1173(2-hydroxy-2-methyl-1-phenyl-1-propane) and 4265 (the combination of2,4,6-trimethylbenzoyldiphenyl-phosphine oxide and2-hydroxy-2-methyl-1-phenyl-propan-1-one); and IRGACURE 784DC(bis(ηsup.5-2,4-cyclopentadien-1-yl)-bis[2,6-difluoro-3-(1H-pyrrol-1-yl)phenyl]titanium).Generally, the amount of photoinitiator (or free radical generators)should be in the range of about 0.1% to about 10% by weight, such asabout 2 to about 6% by weight. The free radical generator concentrationfor benzoin ether is generally from 0.01 to 5% based on the total weightof the curable composition.

A moisture cure catalyst can also be included in an amount effective tocure the composition. For example, from about 0.1 to about 5% by weight,such as about 0.25 to about 2.5% by weight, of the moisture curecatalyst can be used in the invention to facilitate the cure processbeyond that of photo-activated curing. Examples of such catalystsinclude organic compounds of titanium, tin, zirconium and combinationsthereof. Tetraisopropoxytitanate and tetrabutoxytitanate are suitable asmoisture cure catalyst. See also U.S. Pat. No. 4,111,890, the disclosureof which is expressly incorporated herein by reference.

Included in the conventional silicone composition (and other inorganicand organic adhesive polymers) suitable for the invention are variousinorganic fillers. For example, hollow microspheres supplied by Kishunder the trade name Q-CEL are free flowing powders, white in color.Generally, these borosilicate hollow microspheres are promoted asextenders in reactive resin systems, ordinarily to replace heavyfillers, such as calcium carbonate, thereby lowering the weight ofcomposite materials formed therewith. Q-CEL 5019 hollow microspheres areconstructed of a borosilicate, with a liquid displacement density of0.19 g/cm², a mean particle size of 70 microns, and a particle sizerange of 10-150 um. Other Q-CEL products are shown below in tabularform. Another commercially available hollow glass microsphere is sold byKish under the trade name SPHERICEL. SPHEREICEL 110P8 has a meanparticle size of about 11.7 microns, and a crush strength of greaterthan 10,000 psi. Yet other commercially available hollow glassmicrosphere are sold by the Schundler Company, Metuchen, N.J. under thePERLITE tradename, Whitehouse Scientific Ltd., Chester, UK and 3M,Minneapolis, Minn. under the SCOTCHLITE tradename. In general, theseinorganic filler components (and others such as fumed silica) addstructural properties to the cured composition, as well as confersflowability properties to the composition in the uncured state andincrease the transmissivity for the UV cure radiation. When present, thefumed silica can be used at a level of up to about 50 weight percent,with a range of about 4 to at least about 10 weight percent, beingdesirable. While the precise level of silica may vary depending on thecharacteristics of the particular silica and the desired properties ofthe composition and the reaction product thereof, care should beexercised by those persons of ordinary skill in the art to allow for anappropriate level of transmissivity of the inventive compositions topermit a UV cure to occur.

Desirable hydrophobic silicas include hexamethyldisilazane-treatedsilicas, such as those commercially available from Wacker-Chemie,Adrian, Mich. under the trade designation HDK-2000. Others includepolydimethylsiloxane-treated silicas, such as those commerciallyavailable from Cabot Corporation under the trade designation CAB-O-SILN70-TS, or Degussa Corporation under the trade designation AEROSIL R202.Still other silicas include trialkoxyalkyl silane-treated silicas, suchas the trimethoxyoctyl silane-treated silica commercially available fromDegussa under the trade designation AEROSIL R805; and 3-dimethyldichlorosilane-treated silicas commercially available from Degussa underthe trade designation R972, R974 and R976.

While these inorganic fillers have extended the use of conventional UVcured silicone systems to permit the curing of materials beyond a skindepth of UV penetration, these inorganic fillers alone do not overcomeshadowing effects and suffer from UV scattering which effectively makesfor a smaller penetration depth. In the invention described herein, theinclusion of these inorganic fillers along with luminescing particlesprovide a mechanism by which uniform light activated cures can occurdeep inside of the body of adhesive-solidified assemblies in regionsthat would normally be shadowed or not with the reach of external UV orother light sources.

Accordingly, in this example of the invention described herein,conventional silicone and polymeric adhesive or release or coatingcompositions are prepared using conventional mixing, heating, andincubation techniques. Included in these conventional compositions arethe energy modulation structures of this invention (including thediamond or DLC materials). These compositions can then be applied tosurfaces of objects to be fixed together or to surfaces where a hardcoating is desired or cast in a curable form for the production ofmolded objects. These compositions upon activation will produce radiantlight for photoactivated cure of the luminescing particle containingpolymer composition. The density of the energy modulation structures(including the diamond or DLC materials) in these compositions willdepend on the “light transparency” of the luminescing particlecontaining composition. Where these compositions contain a significantamount of the inorganic filler as discussed above, the concentration ofthe upconverter structures can be reduced for example as compared to acomposition with a black color pigment where the light transparency willbe significantly reduced.

U.S. Pat. No. 7,294,656 to Bach et al., the entire disclosure of whichis incorporated herein by reference, describes a non-aqueous compositioncurable by UV radiation broadly containing a mixture of two UV curableurethane acrylates that have several advantages over conventionalradiation-curable compositions. The Bache et al. compositions can becured in a relatively short time using UV-C (200-280 nm), UV-B (280-320nm), UV-A (320-400 nm) and visible (400 nm and above) radiation. Inparticular, Bache et al. compositions can be cured using radiationhaving a wavelength of 320 nm or more. When fully cured (regardless ofthe type of radiation used), the Bach et al. compositions exhibithardnesses and impact resistances at least comparable to conventionalcoatings.

In the invention described here, the energy modulation structures ofthis invention (including the diamond or DLC materials) are added tothese Bach et al. compositions. Due to the fact that the exterior energysource penetrates deeper into the entirety of the Bach et al.compositions, thicker surface coatings can be realized. Further, thecoatings can be applied to intricate surfaces having for example beenprepared with recesses or protrusions.

Moreover, in one embodiment of the invention, an external energy sourceof the initiation energy (x-rays or other high energy source, deep UV,electrons, gamma rays) can be directed to a structural element in whicha gap (or crack) therein was filled with an uncured radiation-curablemedium (such as those described above). The internally generated lightwill cure the uncured radiation-curable medium in the gap (or crack)thereby providing a repair to the structure being irradiated.

Presently, there is available commercial epoxy systems which utilizeepoxy resin injection for the structural restoration of concrete. Epoxyinjection is very often the only alternative to complete replacement ofa structure. It therefore results in great cost savings. Besides fillingthe cracks, epoxy injection is known to protect rebar in the concreteand to stop water leakage. Commercially, the epoxy injection resinprovides a system for welding cracks which restores the originalstrength and loading originally designed into the concrete. Typically,low viscosity resins are pressure injected into the cracks. Often holesare drilled near or into the cracks to provide a conduit for pumping theresin into the cracks.

It, however, takes time for the resin to penetrate into the thinner,even hair line cracks. Unfortunately, time is limited in the presentcommercial systems due to the fact that the resins are premixed withhardeners whose time to cure sets an upper limit for how long the lowviscosity resin can flow into the cracks. Furthermore, time to completerepair is an issue in many industrial repairs as the hardener is usuallypresent in a concentration high enough to have the resin set for examplein twenty four (24) hours. Moreover, with traditional resin methods, itis not possible to induce curing at specific regions of interest sinceall the areas of the resin will be cured

The present invention offers a number of advantages. Firstly, the resinof the present invention will be a photoactivated resin which will notsubstantially cure until the x-ray source generates internal light toactivate the photoinitiators. This provides more flexibility in pumpingand waiting for complete crack fill. Secondly, once the photoactivatableresin is in place, its cure is then activated, and the cure occurs at arate not controlled by the convention hardening reaction. Thirdly, thex-ray penetration through the concrete and the crack region will providea more uniform mechanism for cure of the resins, with the deep cracksbeing as likely to fully cure as the narrow cracks which may extenddeeper into the material. Furthermore, the present invention allows thepossibility to cure only the specific areas of interest, i.e., where theX-ray is irradiated.

In another embodiment of the present invention, the external energysource (x-rays or other high energy source, deep UV, electrons, gammarays) can be a directed or focused beam of the initiation energy whichcures an uncured radiation-curable medium to produce a patternedelement. In this embodiment, the structure holding or at least partiallyenclosing the uncured radiation-curable medium can be a structure opaqueto visible light. In this manner, the uncured radiation-curable medium(which normally would be photoactivated upon exposure to ambient light)can be transported without premature curing. In this embodiment, thecuring would be activated for example by directed one or several focusedbeams of x-rays whose overlap generates regions in the structure holdingor at least partially enclosing the uncured radiation-curable mediumwhere the generated UV or visible light from the energy modulationagents in the medium would be of sufficient intensity to activate thephotoinitiators. In this manner, precise three-dimensional andtwo-dimensional patterning can be performed.

As an example in another embodiment, a patterned element such as adevice (such as plug to close a specific internal hole or path ways) canbe fabricated (e.g., cured) inside structures (e.g., building materials,man-made or natural underground storage tank, internal organs of humanbody, etc) using energy excitation (e.g., X ray) from the outside ofsuch structures. Another application of this technique would involve thefabrication of orthopedic structures inside the body, where the curableresin was introduced locally at the point of the orthopedic structure tobe formed and a directed or focused x-ray beam cured the structure. Inone embodiment, the procedure simultaneously or sequentially sterilizesthe orthopedic structure (or other medical implant) in place. In oneembodiment, the irradiation procedure can be repeated from time to timeto re-sterilize the orthopedic structure (or other medical implantdevices). In one embodiment, the diamond and diamond like converters asused to generate deep UV for sterilization, while other of theabove-described energy modulation generate UV or visible light designedfor polymerization.

Accordingly, in another embodiment of the present invention, there isprovided a method (and associated system) for producing a patternedelement inside a structure. The method places inside the structure aradiation curable medium including at least one of a plasmonics agentand an energy modulation agent such as the diamond or DLC based energymodulation agent. The energy modulation agent is configured to emitlight into the medium upon interaction with an initiation energy. Themethod applies to the medium the initiation energy from a directed orfocused energy source. The applied initiation energy interacts with theplasmonics agent or the energy modulation agent to generate light atlocal regions inside the structure to cure locally the radiation curablemedium.

As noted above, this method can form for the patterned element a plug toclose a hole or pathway in the structure such as for example holes orpathways in a building material, a man-made or natural undergroundstorage tank, or an internal organ in a human or animal body. The methodcan form for the patterned element a prosthetic device at a local pointin the body of a human or animal.

Additional modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

The invention claimed is:
 1. A method for effecting a predeterminedchange in biological activity to a target structure, comprising:providing within close proximity to or within the target structure oneor more light emitters, each capable of emitting at least two differentwavelengths of light, each wavelength of light associated with adifferent biological response, wherein the one or more light emitterscomprise at least one energy modulation agent which adsorbs, intensifiesor modifies an applied initiation energy into an energy that effects thepredetermined change in said target structure; applying the initiationenergy from at least one source to the target structure, activatingplural biological responses in the target structure depending on thedifferent wavelengths of light provided internally within the targetstructure by the one or more light emitters, wherein the differentwavelengths activate different biological responses simultaneously. 2.The method of claim 1, wherein activating plural biological responsescomprises activating a first biological response with a first wavelengthof light and a second biological response with a second wavelength oflight.
 3. The method of claim 2, wherein activating the first biologicalresponse comprises activating psoralen.
 4. The method of claim 3,wherein activating the second biological response comprises generating areactive oxygen species.
 5. The method of claim 2, wherein activatingthe first biological response comprises at least one of photoactivatinga drug, sterilizing the target structure, photoactivating psoralen,generating a reactive oxygen species, inducing an autoimmune response,exciting a DNA strand of a cancer cell, redirecting a metabolic pathway,up-regulating genes, down-regulating genes, secreting cytokines,altering cytokine receptor responses, releasing metabolites, orcombinations thereof.
 6. The method of claim 2, wherein activating thesecond biological response comprises at least one of photoactivating adrug, sterilizing the target structure, photoactivating psoralen,generating a reactive oxygen species, inducing an autoimmune response,exciting a DNA strand of a cancer cell, redirecting a metabolic pathway,up-regulating genes, down-regulating genes, secreting cytokines,altering cytokine receptor responses, releasing metabolites, orcombinations thereof.
 7. The method of claim 2, wherein activating thefirst biological response comprises bonding a pharmaceutical agent to acellular structure.
 8. The method of claim 7, wherein the bondingcomprises bonding the pharmaceutical agent to at least one of nuclearDNA, mRNA, rRNA, ribosome, or mitochondrial DNA.
 9. The method of claim2, wherein at least one of said activating the first biological responseand said activating the second biological response comprises altering acellular response or a metabolic rate of the target structure.
 10. Themethod of claim 2, wherein activating the first biological responsecomprises emitting ultraviolet light to act as a germicide, and whereinactivating the second biological response comprises emitting nearinfrared light to act as an anti-inflammatory.
 11. The method of claim2, wherein activating the first biological response comprises emittingultraviolet light to act as a germicide, and wherein activating thesecond biological response comprises emitting near infrared light topromote cellular proliferation.
 12. The method of claim 2, whereinactivating the first biological response comprises emitting ultravioletlight to act as a germicide, and wherein activating the secondbiological response comprises emitting near infrared light to reducepain.
 13. The method of claim 2, wherein activating the first biologicalresponse comprises photoactivating a pharmaceutical agent, and whereinactivating the second biological response comprises heating a local areaof the target structure.
 14. The method of claim 1, wherein thepredetermined change modifies the target structure or modulates thebiological activity of the target structure.
 15. The method of claim 1,further comprising contacting the target structure with at least oneactivatable pharmaceutical agent (PA) that is capable of effecting saidpredetermined change in the target structure when activated by one ormore of the different wavelengths of light provided internally withinthe target structure.
 16. The method of claim 1, wherein said at leastone energy modulation agent comprises at least one of a biocompatiblefluorescing metal nanoparticle, fluorescing metal oxide nanoparticle,fluorescing metal coated metal oxide nanoparticle, fluorescing dyemolecule, gold nanoparticle, silver nanoparticle, gold-coated silvernanoparticle, a water soluble quantum dot encapsulated by polyamidoaminedendrimers, a luciferase, a biocompatible phosphorescent molecule, acombined electromagnetic energy harvester molecule, and a lanthanidechelate exhibiting intense luminescence.
 17. The method of claim 1,wherein said at least one energy modulation agent decreases a wavelengthof the initiation energy.
 18. The method of claim 17, wherein said atleast one energy modulation agent comprises inorganic materials selectedfrom the group consisting of: metal oxides; metal sulfides; doped metaloxides; and mixed metal chalcogenides.
 19. The method of claim 17,wherein said at least one energy modulation agent comprises at least onemember selected from the group consisting of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄,YAG, YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃,Na-doped YbF₃, ZnS; ZnSe; MgS; CaS, and alkali lead silicatecompositions which contain one or more of SiO₂, B₂O₃, Na₂O, K₂O, PbO,MgO, or Ag.
 20. The method of claim 17, wherein said at least one energymodulation agent comprises at least one of ZnSeS:Cu, Ag, Ce, Tb; CaS:Ce, Sm; La₂O₂S:Tb; Y₂O₂S:Tb; Gd₂O₂S:Pr, Ce, F; LaPO₄.
 21. The method ofclaim 17, wherein said at least one energy modulation agent comprises atleast one of ZnS:Ag, ZnS:Cu, Pb, and alloys of the ZnSeS.
 22. The methodof claim 17, wherein said at least one energy modulation agent comprisesat least one of sodium yttrium fluoride (NaYF₄), lanthanum fluoride(LaF₃), lanthanum oxysulfide (La₂O₂S), yttrium oxysulfide (Y₂O₂S),yttrium fluoride (YF₃), yttrium gallate, yttrium aluminum garnet (YAG),gadolinium fluoride (GdF₃), barium yttrium fluoride (BaYF₅, BaY₂F₈),gadolinium oxysulfide (Gd₂O₂S), calcium tungstate (CaWO₄), yttriumoxide:terbium (Yt₂O₃Tb), gadolinium oxysulphide:europium (Gd₂O₂S:Eu),lanthanum oxysulphide:europium (La₂O₂S:Eu), and gadoliniumoxysulphide:promethium, cerium, fluorine (Gd₂O₂S:Pr,Ce,F), YPO₄:Nd,LaPO₄:Pr, (Ca,Mg)SO₄:Pb, YBO₃:Pr, Y₂SiO₅:Pr, Y₂Si₂O₇:Pr,SrLi₂SiO₄:Pr,Na, and CaLi₂SiO₄:Pr.
 23. The method of claim 17, whereinsaid at least one energy modulation agent comprises at least one ofKSrPO₄:Eu²⁺, Pr³⁺, NaGdF₄:Eu, Zn₂SiO₄:Tb³⁺, Yb³⁺, β-NaGdF₄ co-doped withCe³⁺ and Tb³⁺ ions, and Gd₂O₂S:Tm or BaYF₅:Eu³⁺.
 24. The method of claim1, wherein said at least one energy modulation agent increases awavelength of the initiation energy.
 25. The method of claim 24, whereinsaid at least one energy modulation agent at least one of Y₂O₃, Y₂O₂S,NaYF₄, NaYbF₄, YAG, YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄,YbF₃, YF₃, Na-doped YbF₃, or SiO₂ or alloys or layers thereof.
 26. Themethod of claim 1, wherein said predetermined change results indestruction, lysis or inactivation of the target structure.
 27. Themethod of claim 1, wherein said predetermined change does not result indestruction or inactivation of the target structure.
 28. The method ofclaim 1, wherein said predetermined change enhances an activity of thetarget structure.
 29. The method of claim 1, wherein the targetstructure is a eukaryotic cell.
 30. The method of claim 1, wherein thetarget structure is a prokaryotic cell.
 31. The method of claim 1,wherein the target structure is a subcellular structure.
 32. The methodof claim 31, wherein the subcellular structure is a cell membrane, anuclear membrane, cell nucleus, nucleic acid, mitochondria, ribosome, orother cellular organelle or component.
 33. The method of claim 1,wherein the target structure is an extracellular structure.
 34. Themethod of claim 1, wherein the target structure is a virus or prion. 35.The method of claim 1, wherein the target structure is a cellulartissue.
 36. The method of claim 1, wherein the predetermined changeresults in treatment of a condition, disorder or disease in saidsubject.
 37. The method of claim 36, wherein said condition, disorder ordisease is cancer.
 38. The method of claim 36, wherein said condition,disorder or disease occurs in a soft tissue and/or cartilage.
 39. Themethod of claim 36, wherein said condition, disorder or disease occursin bone tissue.
 40. The method of claim 36, wherein said condition,disorder or disease is chronic pain.
 41. The method of claim 36, whereinsaid condition, disorder or disease is at least one autoimmune disease.42. The method of claim 36, wherein said predetermined change compriseswound healing.
 43. The method of claim 36, wherein said predeterminedchange comprises enhancement of tissue growth, nerve regeneration orsensory regeneration/restoration.
 44. The method of claim 36, whereinsaid condition, disorder or disease is a prion, viral, bacterial,fungal, or parasitic infection.
 45. The method of claim 36, wherein saidpredetermined change comprises reduction or removal of fat deposits. 46.The method of claim 36, wherein said condition, disorder, or disease ischaracterized by varicose veins.
 47. The method of claim 36, whereinsaid condition, disorder, or disease is characterized by an enlargedprostate.
 48. The method of claim 36, wherein said condition, disorder,or disease is characterized by retinal injuries and other oculardiseases.
 49. The method of claim 36, wherein said condition, disorder,or disease is Parkinson's disease.
 50. The method of claim 36, whereinsaid condition, disorder, or disease is characterized by a behavioral,perceptional and/or cognitive disorder.
 51. The method of claim 1,wherein said predetermined change comprises stimulation or modulation ofbrain cell activity with light.
 52. The method of claim 1, wherein saidpredetermined change comprises modulation of cell death.
 53. The methodof claim 1, wherein said predetermined change comprises modulating cellgrowth and division.
 54. The method of claim 1, wherein saidpredetermined change comprises modulation of an activity, quantity, ornumber of intracellular components in a cell.
 55. The method of claim 1,wherein said predetermined change comprises modulation of an activity,quantity, or number of extracellular components produced by, excretedby, or associated with a cell.
 56. The method of claim 1, wherein theinitiation energy is UV radiation, visible light, IR radiation, x-rays,gamma rays, an electron beam, microwaves or radio waves.
 57. The methodof claim 1, wherein the different wavelengths of the light providedinternally within the subject are generated in-situ in the subject. 58.The method of claim 57, wherein the different wavelengths of the lightprovided internally within the subject are generated from a distal endof a catheter inserted into the subject.
 59. The method of claim 57,wherein the different wavelengths of the light provided internallywithin the subject are generated from energy modulation agents disposedat the distal end of the catheter.
 60. The method of claim 57, whereinthe different wavelengths of the light provided internally within thesubject are generated from down converting materials disposed at thedistal end of the catheter.
 61. The method of claim 57, wherein thedifferent wavelengths of the light provided internally within thesubject are generated from up converting materials disposed at thedistal end of the catheter.
 62. The method of claim 1, wherein aplasmonics-active agent is further applied which enhances or modifiesthe initiation energy, such that enhanced initiation energy causes thepredetermined change in said target structure by the plasmonics-activeagent with or without an energy modulation agent.
 63. The method ofclaim 1, wherein a plasmonics-active agent is further applied whichenhances or modifies the initiation energy, such that enhancedinitiation energy is absorbed, intensified or modified by an energymodulation agent into the energy that affects the predetermined changein said target structure.
 64. The method of claim 1, providing at leastone plasmonics-active agent within close proximity to or within thetarget structure.
 65. The method of claim 64, wherein theplasmonics-active comprises plasmonics-active metal nanostructures. 66.The method of claim 65, wherein the metal nanostructures arenanospheres, nanorods, nanocubes, nanopyramids, nanoshells, multi-layernanoshells and combinations thereof.
 67. The method of claim 1, whereinsaid predetermined change modifies the target structure and modulatesbiological activity of the target structure thus treating a condition,disorder or disease affecting the target structure.
 68. The method ofclaim 67, wherein said condition, disorder, or disease is mediated byabnormal cellular proliferation and said predetermined changeameliorates the abnormal cellular proliferation.
 69. The method of claim68, wherein said abnormal cellular proliferation is higher than that ofcells from a subject not having said condition, disorder or disease. 70.The method of claim 68, wherein said abnormal cellular proliferation islower than that of cells from a subject not having said condition,disorder or disease.
 71. The method of claim 67, wherein said condition,disorder, or disease is not significantly mediated by abnormal cellularproliferation and said predetermined change does not substantiallyaffect cellular proliferation.
 72. The method of claim 67, wherein theinitiation energy is x-rays, gamma rays, an electron beam, UV radiation,visible light, or infrared radiation.
 73. The method of claim 67,wherein the initiation energy has the capability of penetratingcompletely through the subject.
 74. The method of claim 67, wherein thecondition, disorder, or disease is a cell proliferation disorder that isat least one member selected from the group consisting of cancer,bacterial infection, viral infection, parasitic infection, prioninfection, fungal infection, immune rejection response, autoimmunedisorders, aplastic conditions, and combinations thereof.
 75. The methodof claim 67, wherein said condition, disorder, or disease is selectedfrom the group consisting of cardiac ablasion, photoangioplasticconditions, intimal hyperplasia, arteriovenous fistula, maculardegeneration, psoriasis, acne, hopecia areata, portwine spots, hairremoval, autoimmune diseases, rheumatoid and inflammatory arthritis,behavioral and cognitive disorders/conditions, joint conditions,Parkinson's disease, retinal injuries and other ocular diseases,enlarged prostate, varicose veins, reduction or removal of fat deposits,nerve regeneration, sensory regeneration/restoration, wound healing,chronic pain, conditions occurring in bone tissue, conditions occurringin a soft tissue and/or cartilage, and lymph node conditions.
 76. Themethod of claim 67, wherein activating comprises activating apharmaceutical agent which is photoactivatable.
 77. The method of claim76, wherein the pharmaceutical agent is selected from psoralens, pyrenecholesteryloleate, acridine, porphyrin, fluorescein, rhodamine,16-diazorcortisone, ethidium, transition metal complexes of bleomycin,transition metal complexes of deglycobleomycin organoplatinum complexes,alloxazines, vitamin Ks, vitamin L, vitamin metabolites, vitaminprecursors, naphthoquinones, naphthalenes, naphthols and derivativesthereof having planar molecular conformations, porphorinporphyrins, dyesand phenothiazine derivatives, coumarins, quinolones, quinones, andanthroquinones.
 78. The method of claim 76, wherein the pharmaceuticalagent is a psoralen, a coumarin, a porphyrin or a derivative thereof.79. The method of claim 76, wherein the pharmaceutical agent is 8-MOP orAMT.
 80. The method of claim 76, wherein the pharmaceutical agent is oneselected from 7,8-dimethyl-10-ribityl, isoalloxazine,7,8,10-trimethylisoalloxazine, 7,8-dimethylalloxazine,isoalloxazine-adenine dinucleotide, alloxazine mononucleotide, aluminum(III) phthalocyanine tetrasulonate, hematophorphyrin, andphthadocyanine.
 81. The method of claim 76, wherein the pharmaceuticalagent is coupled to a carrier that is capable of binding to a receptorsite on or near the target structure.
 82. The method of claim 81,wherein the carrier is one selected from insulin, interleukin,thymopoietin or transferrin.
 83. The method of claim 81, wherein thepharmaceutical agent is coupled to the carrier by a covalent bond. 84.The method of claim 81, wherein the pharmaceutical agent is coupled tothe carrier by non-covalent bond.
 85. The method of claim 81, whereinthe receptor site is one selected from nucleic acids of nucleated cells,antigenic sites on nucleated cells, or epitopes.
 86. The method of claim76, wherein the pharmaceutical agent has an affinity for the targetstructure.
 87. The method of claim 76, wherein the target structure is atarget cell and the at least one activatable pharmaceutical agent iscapable of being preferentially absorbed by the target cell.
 88. Themethod of claim 76, wherein the target structure is a target cell andthe predetermined change is apoptosis in the target cell.
 89. The methodof claim 76, wherein the pharmaceutical agent, upon activation, causesan auto-vaccine effect in the subject that reacts with the targetstructure.
 90. The method of claim 76, wherein the pharmaceutical agentis a DNA intercalator or a halogenated derivative thereof.
 91. Themethod of claim 76, wherein the pharmaceutical agent comprises alight-sensitive protein that upon exposure to said initiation energysource modulates a signaling event in the brain.
 92. The method of claim76, wherein said predetermined change enhances the expression of,promotes the growth of, or increases the quantity of said targetstructure.
 93. The method of claim 76, wherein said predetermined changeenhances, inhibits or stabilizes the usual biological activity of saidtarget structure compared to a similar untreated target structure. 94.The method of claim 76, wherein said predetermined change alters theimmunological or chemical properties of said target structure.
 95. Themethod of claim 94, wherein said target structure is a compound that ismodified by said predetermined change to be more or less antigenic orimmunogenic.
 96. The method of claim 1, wherein the one or more lightemitters comprise at least one of a diamond light emitter or adiamond-like carbon light emitter.
 97. The method of claim 1, whereinthe one or more light emitters comprise one or more coated energymodulation agents.
 98. The method of claim 1, wherein the one or morecoated energy modulation agents have a biocompatible coating.
 99. Themethod of claim 1, wherein the biocompatible coating comprises at leastone of poly(esters) based on polylactide (PLA), polyglycolide (PGA),polycarpolactone (PCL), poly(hydroxyalkanoate)s of the PHB-PHV class,poly(ester)s, natural polymers, modified poly(saccharide)s, starch,cellulose, chitosan, polyethylene oxides, poly(ether)(ester) blockcopolymers, and ethylene vinyl acetate copolymers.
 100. The method ofclaim 1, wherein the biocompatible coating comprises at least one of asilica, a silicate, nano-diamond film, a diamond like carbon coating, ora graphene material.
 101. A method for effecting a predetermined changein biological activity to a target structure, comprising: providingwithin close proximity to or within the target structure one or morediamond or diamond-like carbon light emitters capable, upon exposure toan applied initiation energy, of emitting one or more wavelengths oflight corresponding to respective one or more biological responses;applying the initiation energy from at least one source to the targetstructure, thereby activating the one or more biological responses inthe target structure depending on the emitted one or more wavelengths oflight provided internally within the target structure from the one ormore diamond or diamond-like carbon light emitters.
 102. The method ofclaim 101, wherein the initiation energy contacts the target structure,activates the one or more biological responses, and induces thepredetermined change in the target structure.
 103. The method of claim101, wherein the activating biological responses comprises at least oneof 1) activating a first biological response with a first wavelength oflight and 2) activating a second biological response with a secondwavelength of light.
 104. The method of claim 103, wherein activatingthe first biological response comprises activating psoralen.
 105. Themethod of claim 104, wherein activating the second biological responsecomprises generating a reactive oxygen species.
 106. The method of claim103, wherein activating the first biological response comprises at leastone of photoactivating a drug, sterilizing the target structure,photoactivating psoralen, generating a reactive oxygen species, inducingan autoimmune response, exciting a DNA strand of a cancer cell,redirecting a metabolic pathway, up-regulating genes, down-regulatinggenes, secreting cytokines, altering cytokine receptor responses,releasing metabolites, or combinations thereof.
 107. The method of claim103, wherein activating the second biological response comprises atleast one of photoactivating a drug, sterilizing the target structure,photoactivating psoralen, generating a reactive oxygen species, inducingan autoimmune response, exciting a DNA strand of a cancer cell,redirecting a metabolic pathway, up-regulating genes, down-regulatinggenes, secreting cytokines, altering cytokine receptor responses,releasing metabolites, or combinations thereof.
 108. The method of claim103, wherein activating the first biological response comprises bondinga pharmaceutical agent to a cellular structure.
 109. The method of claim108, wherein the bonding comprises bonding the pharmaceutical agent toat least one of nuclear DNA, mRNA, rRNA, ribosome, or mitochondrial DNA.110. The method of claim 103, wherein at least one of said activatingthe first biological response and said activating a second biologicalresponse comprises altering a cellular response or a metabolic rate ofthe target structure.
 111. The method of claim 103, wherein activatingthe first biological response comprises emitting ultraviolet light toact as a germicide, and wherein activating the second biologicalresponse comprises emitting near infrared light to act as ananti-inflammatory.
 112. The method of claim 103, wherein activating thefirst biological response comprises emitting ultraviolet light to act asa germicide, and wherein activating the second biological responsecomprises emitting near infrared light to promote cellularproliferation.
 113. The method of claim 103, wherein activating thefirst biological response comprises emitting ultraviolet light to act asa germicide, and wherein activating the second biological responsecomprises emitting near infrared light to reduce pain.
 114. The methodof claim 103, wherein activating the first biological response comprisesphotactivating a pharmaceutical agent, and wherein activating the secondbiological response comprises heating a local area of the targetstructure.
 115. The method of claim 101, wherein the predeterminedchange modifies the target structure or modulates the biologicalactivity of the target structure.
 116. The method of claim 101, furthercomprising contacting the target structure with at least one activatablepharmaceutical agent (PA) that is capable of effecting saidpredetermined change in the target structure when activated thedifferent wavelengths of light provided internally within the targetstructure.
 117. The method of claim 101, wherein said initiation energygenerates light that induces the predetermined change in the targetstructure with or without an energy modulator or a photoactive agent.118. The method of claim 101, further comprising administering to saidsubject at least one energy modulation agent which adsorbs, intensifiesor modifies said initiation energy into an energy that in conjunctionwith light from the one or more diamond or diamond-like carbon lightemitters effects the predetermined change in said target structure. 119.The method of claim 118, wherein said at least one energy modulationagent comprises at least one of a biocompatible fluorescing metalnanoparticle, fluorescing metal oxide nanoparticle, fluorescing metalcoated metal oxide nanoparticle, fluorescing dye molecule, goldnanoparticle, silver nanoparticle, gold-coated silver nanoparticle, awater soluble quantum dot encapsulated by polyamidoamine dendrimers, aluciferase, a biocompatible phosphorescent molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelateexhibiting intense luminescence.
 120. The method of claim 118, whereinsaid at least one energy modulation agent decreases a wavelength of theinitiation energy.
 121. The method of claim 120, wherein said at leastone energy modulation agent comprises inorganic materials selected fromthe group consisting of: metal oxides; metal sulfides; doped metaloxides; and mixed metal chalcogenides.
 122. The method of claim 120,wherein said at least one energy modulation agent comprises at least onemember selected from the group consisting of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄,YAG, YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃,Na-doped YbF₃, ZnS; ZnSe; MgS; CaS, and alkali lead silicatecompositions which contain one or more of SiO₂, B₂O₃, Na₂O, K₂O, PbO,MgO, or Ag.
 123. The method of claim 120, wherein said at least oneenergy modulation agent comprises at least one of ZnSeS:Cu, Ag, Ce, Tb;CaS: Ce,Sm; La₂O₂S:Tb; Y₂O₂S:Tb; Gd₂O₂S:Pr, Ce, F; LaPO₄.
 124. Themethod of claim 120, wherein said at least one energy modulation agentcomprises at least one of ZnS:Ag, ZnS:Cu, Pb, and alloys of the ZnSeS.125. The method of claim 120, wherein said at least one energymodulation agent comprises at least one of sodium yttrium fluoride(NaYF₄), lanthanum fluoride (LaF₃), lanthanum oxysulfide (La₂O₂S),yttrium oxysulfide (Y₂O₂S), yttrium fluoride (YF₃), yttrium gallate,yttrium aluminum garnet (YAG), gadolinium fluoride (GdF₃), bariumyttrium fluoride (BaYF₅, BaY₂F₈), gadolinium oxysulfide (Gd₂O₂S),calcium tungstate (CaWO₄), yttrium oxide:terbium (Yt₂O₃Tb), gadoliniumoxysulphide:europium (Gd₂O₂S:Eu), lanthanum oxysulphide:europium(La₂O₂S:Eu), and gadolinium oxysulphide:promethium, cerium, fluorine(Gd₂O₂S:Pr,Ce,F), YPO₄:Nd, LaPO₄:Pr, (Ca,Mg)SO₄:Pb, YBO₃:Pr, Y₂SiO₅:Pr,Y₂Si₂O₇:Pr, SrLi₂SiO₄:Pr,Na, and CaLi₂SiO₄:Pr.
 126. The method of claim120, wherein said at least one energy modulation agent comprises atleast one of KSrPO₄:Eu²⁺, Pr³⁺, NaGdF₄:Eu, Zn₂SiO₄:Tb³⁺, Yb³⁺, β-NaGdF₄co-doped with Ce³⁺ and Tb³⁺ ions, and Gd₂O₂S:Tm or BaYF₅:Eu³⁺.
 127. Themethod of claim 118, wherein said at least one energy modulation agentincreases a wavelength of the initiation energy.
 128. The method ofclaim 127, wherein said at least one energy modulation agent at leastone of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄, YAG, YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃,TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, Na-doped YbF₃, or SiO₂ or alloys or layersthereof.
 129. The method of claim 101, wherein said predetermined changeresults in destruction, lysis or inactivation of the target structure.130. The method of claim 101, wherein said predetermined change does notresult in destruction or inactivation of the target structure.
 131. Themethod of claim 101, wherein said predetermined change enhances anactivity of the target structure.
 132. The method of claim 101, whereinthe target structure is a eukaryotic cell.
 133. The method of claim 101,wherein the target structure is a prokaryotic cell.
 134. The method ofclaim 101, wherein the target structure is a subcellular structure. 135.The method of claim 134, wherein the subcellular structure is a cellmembrane, a nuclear membrane, cell nucleus, nucleic acid, mitochondria,ribosome, or other cellular organelle or component.
 136. The method ofclaim 101, wherein the target structure is an extracellular structure.137. The method of claim 101, wherein the target structure is a virus orprion.
 138. The method of claim 101, wherein the target structure is acellular tissue.
 139. The method of claim 101, wherein the predeterminedchange results in treatment of a condition, disorder or disease in saidsubject.
 140. The method of claim 139, wherein said condition, disorderor disease is cancer.
 141. The method of claim 139, wherein saidcondition, disorder or disease occurs in a soft tissue and/or cartilage.142. The method of claim 139, wherein said condition, disorder ordisease occurs in bone tissue.
 143. The method of claim 139, whereinsaid condition, disorder or disease is chronic pain.
 144. The method ofclaim 139, wherein said condition, disorder or disease is at least oneautoimmune disease.
 145. The method of claim 139, wherein saidpredetermined change comprises wound healing.
 146. The method of claim139, wherein said predetermined change comprises enhancement of tissuegrowth, nerve regeneration or sensory regeneration/restoration.
 147. Themethod of claim 139, wherein said condition, disorder or disease is aprion, viral, bacterial, fungal, or parasitic infection.
 148. The methodof claim 139, wherein said predetermined change comprises reduction orremoval of fat deposits.
 149. The method of claim 139, wherein saidcondition, disorder, or disease is characterized by varicose veins. 150.The method of claim 139, wherein said condition, disorder, or disease ischaracterized by an enlarged prostate.
 151. The method of claim 139,wherein said condition, disorder, or disease is characterized by retinalinjuries and other ocular diseases.
 152. The method of claim 139,wherein said condition, disorder, or disease is Parkinson's disease.153. The method of claim 139, wherein said condition, disorder, ordisease is characterized by a behavioral, perceptional and/or cognitivedisorder.
 154. The method of claim 101, wherein said predeterminedchange comprises stimulation or modulation of brain cell activity withlight.
 155. The method of claim 101, wherein said predetermined changecomprises modulation of cell death.
 156. The method of claim 101,wherein said predetermined change comprises modulating cell growth anddivision.
 157. The method of claim 101, wherein said predeterminedchange comprises modulation of an activity, quantity, or number ofintracellular components in a cell.
 158. The method of claim 101,wherein said predetermined change comprises modulation of an activity,quantity, or number of extracellular components produced by, excretedby, or associated with a cell.
 159. The method of claim 118, whereinsaid one or more diamond or diamond-like carbon light emitters areprovided as a coating on at least a portion of a surface of the energymodulation agent.
 160. The method of claim 159, wherein said one or morediamond or diamond-like carbon light emitters form a coating on all ofthe surface of the energy modulation agent.
 161. The method of claim101, wherein said one or more diamond or diamond-like carbon lightemitters comprise a plurality of defects in the diamond or diamond-likecarbon light emitter such that upon excitation, a plurality ofwavelengths of light are emitted.
 162. The method of claim 161, whereinsaid one or more diamond or diamond-like carbon light emitters is asingle diamond or diamond-like carbon light emitter having the pluralityof defects present.
 163. The method of claim 161, wherein said one ormore diamond or diamond-like carbon light emitters are a plurality oftypes of diamond or diamond-like carbon light emitters each of theplurality of types has a different defect than the others of theplurality of types.